Team:Cambridge/Bacillus/Lab Work

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=July 22nd=
=July 22nd=
Line 230: Line 245:
Even with a concentration of 35μg/mL, ther is not a clear area around the antibiotic disk. So we have to test some higher concentration of Cm.
Even with a concentration of 35μg/mL, ther is not a clear area around the antibiotic disk. So we have to test some higher concentration of Cm.
-
=Wet Work=
+
==Wet Work==
* New antibiotic tests
* New antibiotic tests
Line 264: Line 279:
Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them!
Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them!
-
 
=July 28th=
=July 28th=
Line 811: Line 825:
=August 6th=
=August 6th=
-
==PCR parts of Agr operon==
+
==Result from transformation==
 +
PLATES HAD BEEN BINNED!! OOPS
-
We received primers for Agr A,B,C, and D today - http://openwetware.org/wiki/IGEM:Cambridge/2008/Turing_Pattern_Formation/Primers
+
==Check Vectors==
-
We must PCR each individual part out of the previous BioBricks (I746001 &I746101) in order to put our bacillus RBS on them.
+
- Plasmid miniprep for ECE176 (from 05/08 LB stock and from 04/08 LB stock with a second inoculation)
-
I was concerned that the region of most homology would be the BioBrick prefix and suffix itself, so I first digested the DNA with Xbal and SpeI.  
+
We want to check uncut plasmid, single and double digest for ECE147, ECE149, ECE150, ECE153, ECE162, ECE172, ECE176. In order to have enough DNA (to see the bands), we will add 1μg of DNA to make single digest, and wr will incubate during 1h (instead of only 10min).
-
For some reason the digestion didn't work that well, so I've decided to go ahead and use the uncut BioBrick DNA as template for my reactions.
+
* Nanodrop
 +
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
!  Vetor !! 260/280 !! μg/mL
 +
|-
 +
| ECE147 || 1.67 || 50.8 
 +
|-
 +
| ECE149 || 1.66 || 51.9
 +
|-
 +
| ECE150 || 1.78 || 107.0 
 +
|-
 +
| ECE153 || 1.68 || 31.2 
 +
|-
 +
| ECE162|| 1.56 || 33.1
 +
|-
 +
| ECE172 (04/08)|| 1.64 || 22.8
 +
|-
-
=August 9th=
 
-
==Plans for Monday==
+
| ECE172 (05/08) || 1.89 || 213.1
-
===Lab Work===
+
|-
-
*Be sure that the Spc50 plates are working, as the control for Spc50 grew and shouldn't have. We already know that things can die at Spc100 (the double crossover test from earlier this week) so it is unlikely that the stock is bad. We should test Spc50 again with regular bacillus and with E. coli at 50-100 ug/ml and hope that they die.
+
| ECE176 (04/08)|| 1.89 || 111.1
-
*Finish all the agr PCRs, but especially the RBS.
+
|-
-
*Confirm that amyE insertion works, using the amylase/ery tests.
+
| ECE176 (05/08) || 1.83 || 96.2
-
*Confirm that with ECE153 inserted,  we get detectable fluoresence in a microscope.
+
|}
-
*Confirm that ECE166 transformation works, using plasmid miniprepping.
+
-
**Find the qiagen miniprep kit or adapt the protocol.
+
-
*Confirm that when ECE166 is transformed, we get detectable fluoresence in a microscope.
+
-
===Book/Computer Work===
+
-
*Find good promoters in vectors and order PCR primers to get them out
+
-
*Find out how to assay for/make AIP
+
-
**Find/Contact Jim Ajioka
+
-
*Find out how to do a c-terminal GFP fusion for membrane proteins agrB and agrC
+
-
**Can we fuse GFP and still expect them to localize to the membrane?
+
-
***AgrB transmembrane topology (http://www.jbc.org/cgi/content/full/277/38/34736#F1). The signal peptide is not on the N terminus, and both the N and C terminus are intracellular.
+
-
***AgrC transmembrane topology (http://openurl.ingenta.com/content?genre=article&issn=0950-382X&volume=28&issue=3&spage=655&epage=662 (see figure 2 B)), the N terminus is probably extracellular.  The C terminus is probably intracellular.  The article doesn't say where the signal peptide is.
+
-
**Should we do a antibody tag instead?
+
-
==Longer Term Plans==
+
We made singe digest for the samples from 05/08 only.
-
*Ligate the RBS into a BB vector so we can build a RBS/GFP construct.
+
-
*Put the RBS/GFP construct into ECE166 and ECE 153.
+
-
*Compare the 'out of the box' ECE153 with ECE153 with RBS/GFP biobrick inserts
+
-
*Compare the 'out of the box' ECE166 with ECE166 with RBS/GFP biobrick inserts
+
 +
- Double digest (1 hour of incubation in water bath at 37°C)
-
=August 11th=
+
- Gel
-
==PCR Bacillus RBS==
+
-
We created 2 different Bacillus RBS sites simply by primer annealing and extension. Each product includes an 8bp RBS plus the BioBrick prefix and suffix for a total of 54bp.
+
Gel1
-
The products were visualized on a 3.5% Agarose gel with bioline's hyperladder V.
+
* Lane1 : HyperladderI
 +
* Lane2 : ECE147 with EcoRI
 +
* Lane3 : ECE147 with HindIII
 +
* Lane4 : ECE149 with SpeI
 +
* Lane5 : ECE149 with PstI
 +
* Lane6 : ECE150 with SpeI
 +
* Lane7 : ECE150 with HindIII
 +
* Lane8 : ECE153 with SalI
 +
* Lane9 : ECE162 with SalI
 +
* Lane10 : ECE172 with HindIII
 +
* Lane11 : ECE176 with EcoRI
 +
* Lane12 : HyperladderI
-
RBSs is the consensus RBS sequence for Bacillus subtilis, and thus we believe it to be a very strong binding site -  AAAGGAGG
+
[[Image:photok.gif|300px|center]]
-
RBSw has a 2bp modification from RBSs, which we believe will weaken it -  AGAGGTGG
+
Gel2
 +
* Lane1 : HyperladderI
 +
* Lane2 : ECE176 with XbaI
 +
* Lane3 : ECE176 double digest
 +
* Lane4 : ECE150 double digest
 +
* Lane5 : ECE149 double digest
 +
* Lane6 : ECE147 double digest
 +
* Lane7 : HyperladderI
 +
* Lane8 : ECE149 uncut
 +
* Lane9 : ECE153 uncut
 +
* Lane10 : ECE172 uncut
 +
* Lane11 : supercoiled ladder
 +
[[Image:photol.gif|300px|center]]
-
The amplification of RBSs yielded only one band on the gel. The product was purified using microclean and contained 14.8ng/uL after clean-up.
+
* Results
-
RBSw, however produced 2 bands on the gel. The correct size was gel-extracted and purified. After purification the yield was 12.3 ng/uL.
+
Our supercoiled ladder was very bad, so it was impossible to conclude for uncut vectors.
 +
- ECE147, ECE 150, ECE153, ECE 176 single digest : ok
-
=August 13th=
+
- ECE149 : problem, 2 cutting sites for PstI (only one in the sequence)
-
==PCR Agr A, B, C, D==
+
- ECE162 : only one band whereas SalI should have 2 cutting sites
 +
- ECE172 : wrong sizes!!!
-
Doing a PCR directly from the old Biobricks has worked for all parts of the operon. No prior digestion seems to be needed.
+
- Double digest for ECE176 : ok!
 +
 
 +
- Double digest for ECE 150, 149 and 147 : only one band!!!
 +
 
 +
There may be a problem when we do the double digest by adding two single digest. We will try again with a direct double digest.
 +
 
 +
=August 7th=
 +
 
 +
==Transformation of B.S. IA771==
 +
 
 +
New transformation with the same protocol than 2 days ago. We will try to add less liquid on plates (to avoid growing colonies in liquid)
{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
!  Part !! 260/280 !! μg/mL
+
!  Tube !! Plate 1 !! Plate 2 !! Plate 3 !! Plate 4
|-
|-
-
| A || 1.78 || 92.5
+
| ECE166 || Cm5 + 200μL of cells || Cm5 + 100μL of cells || Cm5 + 50μL of cells || Cm10 + 100μL of cells 
-
|-
+
-
| B || 1.87|| 96.5
+
|-
|-
-
| C || 1.9 || 127.7
+
| ECE153 || Spc50 + 200μL of cells || Spc50 + 100μL of cells || Spc50 + 50μL of cells ||  
|-
|-
-
| D || 1.82 || 26.3
+
| ECE166 || Cm5 + 100μL of cells || Cm10 + 100μL of cells ||  ||
 +
|-
 +
| ECE153 || Spc50 + 100μL of cells ||  ||  ||
 +
|-
 +
| no DNA || Cm5 + 100μL of cells || Cm10 + 100μL of cells || Spc50 + 100μL of cells || 
 +
|-
 +
| ECE166 (glycerol stock)|| Cm5 + 100μL of cells || Cm10 + 100μL of cells ||  ||
|}
|}
 +
==Check Vectors==
-
=August 15th=
+
We want to make a final check for ECE147, 149, 150, 153 and 162.
-
==PCRing out promoters==
+
- Double digest + gel
-
- 4 different promoters : Pxyl, Pspc, Ppac, Pupp
+
* Results
-
- Make master mix enough for 4RXNs
+
* Lane3 : HyperladderI
 +
* Lane4 : ECE147
 +
* Lane5 : ECE149
 +
* Lane6 : ECE150
 +
* Lane7 : ECE153
 +
* Lane8 : ECE162
 +
* Lane9 : HyperladderI
 +
[[Image:photom.gif|300px|center]]
 +
- ECE 147, ECE150, ECE153 :ok!
-
==Digest and Ligate Agr A and C to pSB4C5==
+
- ECE 149 : 3 bands! not the right vector
-
Previous PCR products of Agr A and C were digested using Fermentas Fast Digest - EcoRI and PstI according to their protocol except a longer time was given to digest.
+
- ECE162 : ony one band! It has been uncut... maybe the good vector (according to precedent gel), but not sure
-
3uL of AgrA was used and 4.4 uL of AgrC was used and incubated for 40minutes.
+
==Test ECE112 transformation==
-
10uL totaling to 1ug of pSB4C5 plasmid was also digested and incubated for 10 minutes.
+
- for the three different strain (1A1, IA771, IA751), make 12 spots
-
The digestion was visualized on a 0.8% Agarose gel and produced expected sizes:
+
- take one colony with a loop, and put it on a Cm5 plate and on a Cm5 + Spc100 plate (do that for the 12 spots)
-
Plasmid: approx 3bk
+
- incubate
-
Agr A: approx 700bp
+
==New stock==
-
Agr C: approx 1300bp
+
- Put ECE171 in 10mL of LB
 +
- Incubate
-
=August 16th=
+
=August 8th=
 +
==Result from yesterday transformation==
-
==Agr A and C ligation to pSB4C5==
+
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
!  Vector !! Antibiotic !! Quantity of cells added !! number of colonies
 +
|-
 +
| ECE166 || Cm5 || 200μL || 0 : problem!!! 
 +
|-
 +
| ECE166 || Cm5 || 100μL || 2
 +
|-
 +
| ECE166 || Cm5 || 50μL || 1
 +
|-
 +
| ECE166 || Cm10 || 100μL || 0 : no resistance to Cm10!
 +
|-
 +
| ECE166 (spin) || Cm5 || 100μL || a lot, confluent
 +
|-
 +
| ECE166 (spin) || Cm10 || 100μL || 0
 +
|-
 +
| ECE166 (glycerol + spin) || Cm5 || 100μL || about 150, very small
 +
|-
 +
| ECE166 (spin) || Cm10 || 100μL || 0
 +
|-
 +
| ECE153 || Spc50 || 200μL || 21 + a lot of confluent colonies
 +
|-
 +
|  ECE153 || Spc50 || 100μL || 7 + confluent (a few)
 +
|-
 +
|  ECE153 || Spc50 || 50μL || 4
 +
|-
 +
|  ECE153 (spin) || Spc50 || 100μL || 25 + about 200 (small and almost confluent)
 +
|-
 +
|  No DNA || Cm5 || 100μL || 0
 +
|-
 +
|  No DNA || Cm10 || 100μL || 0
 +
|-
 +
|  No DNA || Spc50 || 100μL || about 12, maybe more : problem!!!!!
 +
|}
-
Ligation was performed using Fermentas Rapid Ligation kit according to their protocol.
+
==Result for test of ECE112 transformation==
-
After ligation, samples were visualized on a gel, but little product of the correct size was seen.  
+
On Cm5 plates, we have some colonies for each strains, on Cm5 + Spc 100 plates, no colonies on each plates. This result seems good, however, we forgot to make a control. Since, we had some colonies on control plates, it is possible that our bacillus are resistant to Cm5, even if they are not transformed.
 +
We addded some control colonies.
-
For agrA we should have gotten a band of size 3700 (3000plasmid + 700 insert)
+
==Control with erythromycin==
-
For agrC we should have gotten a band of size 4300 (3000plasmid + 1300insert)
+
- Prepare erythromycin, stock 5mg/mL : 0.05g in 10mL of SDW
 +
- Prepare plates : add 20μL of Ery (5mg/mL) into 200mL of agar (final concentration : 0.5μg/mL)
-
The bands of appropriate sizes were extracted for transformation.
+
- Do several spots on each plate
 +
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
!  Plate !! Colonies added from
 +
|-
 +
|1 || ECE166 + 100μL of cells (2 different plates) 
 +
|-
 +
|2|| ECE153 + 100μL of cells (2 different plates)
 +
|-
 +
|3 || ECE166 (from glycerol stock)
 +
|-
 +
|4 || Control (from a plate of IA771 without antibiotic)
 +
|-
 +
|5 || IA771 + ECE112 (05/08)
 +
|-
 +
|6 || IA771 + ECE166 (05/08)
 +
|}
 +
<!-- ## Do not edit below this line unless you know what you are doing. ## -->
-
=August 18th=
+
==Amylase production screening==
-
==Check promoters==
+
* Prepare agar plate with starch
-
On Friday, PCR out promoters, we want to check them.
+
- Starch : 1g of starch in 10mL of SDW
-
- Run a gel
+
- Add 2mL of Starch solution into 200mL of agar, mix
-
- Results : good size of bands!!!
+
-Inoculate on different plates : 1A1 +ECE112 (plate from 05/08), IA751 + ECE112 (plate from 05/08) and IA771 + ECE153
-
==Transformation of AgrA and AgrC==
+
==New stocks==
-
AgrA and C gell-extracted ligation was transformed into Top 10 cells using standard protocol. Puc9 was used as a positive control.
+
- Take 1mL from LB stock oh ECE171 (from 07/08) and put it into 99mL of LB
 +
<!-- ## Do not edit below this line unless you know what you are doing. ## -->
 +
=August 11th=
-
=August 19th=
+
==Erytromycin Experiment==
-
==Results of AgrA and C transformation==
+
- New preparation of Ery : 0.05g into 10mL of ETHANOL
-
Transformation has failed. No colonies were visible for the plates of Agr A or C. The Puc9 positive control grew.
+
-
We believe that the problem was in the gel-extraction between ligation and transformation. Most of our plasmid was probably lost in this step. Next time we will directly use the results from the ligation reaction to transform. Although many bands were seen on the previous gel of the ligation reaction, we will check our transformation growth by single colony PCR to confirm transformation of the plasmid with our correct insert.
+
 +
- We plated again our plates (same than 08/08/08)
-
==Lux parts==
+
==Amylase screening experiment==
-
To make the Lux Receiver, we need 4 different parts ;
+
- New method : add 2g of starch powder into 200mL of Agar, shake, pipette to plate (and avoid bubbles)
-
* R0040, TetR repressible promoter
+
- Same plates than 08/08/08
-
* SO168, luxR + double terminator
+
==Test our stock of ECE171==
-
* R0062, promoter activated by luxR
+
-Plasmid miniprep 9from the samples of 10mL and the one of 100mL)
-
* JO4630 (GFP + double terminator)
+
-Nanodrop both DNA
-
All these parts have been transformed into E.coli. We want to test them. R004, R0062 and JO4630 have already been tested, it should be fine.  
+
{|class="wikitable" style="text-align:center" border="1"
-
We received from the MIT R0040, R0062 and S068 already transformed into E.coli, so we want to check these stocks (which are certainly fine) and use them.
+
|-
-
For JO4630, we want to double check our transformation.
+
!  Vetor !! 260/280 !! μg/mL
 +
|-
 +
| ECE171 (10mL) || 1.73 || 136.3
 +
|-
 +
| ECE171(100mL) || 1.82 || 99.5
 +
|}
-
- Plate on antibiotic plates and do LB stocks of single colony from the MIT stock (R0040, R0062 and S0168).
+
- Double digest (EcoRI and PstI) with 10μL of DNA and 1h of incubation
-
- Put on Kan plates 4 different colonies from J04630 (transformation Amp plate) and also incubate these colonies into LB
+
- Samples have been frozen, they should be run onto a gel
 +
==Control of Spc resistance of Bacillus==
-
=August 20th=
+
- Spc50 plate with transformed  IA771 (ECE153, which is Spc resistant) and with IA771 9which should not be Spc resistant)
-
==Check Lux components==
+
=August 12th=
-
- Single colony PCR for :
+
==Results from yesterday==
 +
* Ery plates
-
*R0040 (MIT stuff)
+
- IA751 (control) : most of colonies did not grow, which is good, but 2 or 3 grew...
-
*R0062 (MIT stuff)
+
- IA7771 + ECE166 : all colonies grew : ok 9non integration vector)
-
*4 different colonies of S0168 (from a transformation plate from 12/08)
+
- IA771 + ECE153 : growth of all colonies... problem!!! if IA771 is transformed with this integration vector, it should lose its EryR
-
*4 different colonies of J04630 (from a transformation plate from 12/08)
+
- IA771 + ECE112 : growth of all colonies... problem!!! if IA771 is transformed with this integration vector, it should lose its EryR
-
- Protocol : add 1μL of cells (diluted in water), 10μL of Master Mis, 7μL of SDW, 1μL of VF primer and 1μL of VR primer
 
-
- Gel PCR products
+
* Spc resistance
-
Gel 1
+
- IA771 : no growth on Spc50 plate, so no natural resistance
-
* Lane2 : Hyperladder1
+
- transformed cells grew
-
* Lane3 : JO4630, colony 1
+
-
* Lane4 : JO4630, colony 2
+
-
* Lane5 : JO4630, colony 3
+
-
* Lane6 : JO4630, colony 4
+
-
* Lane7 : HyperladderI
+
-
[[Image:photon.gif|300px|center]]
+
* Amylase plates
-
Gel 2
+
- Add iodine solution for 1min
-
* Lane2 : HyperladderI
+
- Problem : no aparent blue, no diffusion of iodine into LA agar + a lot of contamination
-
* Lane3 : ECE190 double digest
+
-
* Lane4 : S0168, colony 1
+
-
* Lane5 : S0168, colony 2
+
-
* Lane6 : S0168, colony 3
+
-
* Lane7 : S0168, colony 4
+
-
* Lane8 : HyperladderI
+
-
* Lane9 : R0040
+
-
* Lane10 : R0062
+
-
* Lane11 : Ladder 100bp
+
-
[[Image:photoo.gif|300px|center]]
+
==New plates and LB stocks==
-
- '''Results'''
+
- In order to try to make plasmid miniprep at the end of the day, grow IA771 transformed with ECE166 in 10mL LB + Cm5
-
* R0040 and R0062 : one big band of about 300b (expected size 293), OK!
+
7hours is not enough to reach exponential phase!!! We will try again tomorrow by incubating longer
-
* S0168 : one band of about 400b for the 4 different colonies (expected size 1234!), bad! This plate does not contain S0168
+
- Grow IA771 and IA751 ( first plate from sterile disks of strain) imto 10mL of LB
-
* J04630 (colonies 2 and 4) : one band of about 1100b (expected size 1173), OK!
+
- Grow ECE166, 171, 153 in E.coli with approriate antibiotics 9to transform tomorrow)
-
* J04630 (colony 1) : one good band plus another band...
+
- Grow IA771 transformed with ECE153 into LB+Spc50 (to check fluorescence with xylose induction)
-
* J04630 (colony 3) : one band of about 600b, bad!
 
 +
==New test for amylase==
 +
We tried 2 different methods.
-
==Ligation==
+
- Dilute 1g of starch into 100mL of agar and try to dilute it and boil it to sterilize. The problem is that it is very difficult to dilute...
-
- Materials :
+
- The second method seem to be better. We diluted 1g of starch into 100mL of Soft Agar (it dilutes very well). Then plate blank agar plate (LA) and then add a thin layer of SA ith 1% of starch. Poke the plates.
-
*AgrA
+
- We plated IAI+ECE112, IA751+ECE112 and also IA751 and 1A1 for control.
-
*AgrB
+
-
*AgrC
+
-
*AgrD
+
-
*Pupp
+
-
*Pspac
+
-
*Ppac
+
-
*Pxyl
+
-
*RBS S
+
-
*RBS W
+
-
*psB4C5
+
-
- Double digest of PCR products
+
=August 13th=
-
- Run vector, AgrA and AgrD on a gel
+
==Results for Starch plates==
-
- DNA clean and concentrator for AgrA, B,C and D, promoters
+
- Add 5mL of iodine
-
- Microclean for both RBS
+
- 1A1 or IA751 : big zones of clearance
-
- Nanodrop
+
- IA751 + ECE112 : no zones of clearance (photos), just small white points on colonies : the gene AmyE seems to be knocked out, transformation ok!
 +
 
 +
- 1A1 + ECE112 : problem, soft agar melted... impossible to observe!
 +
 
 +
 
 +
==Transformation of IA751==
 +
 
 +
- We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171
 +
 
 +
- Spectrophotometer : blank made with medium A
{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
! !! 260/280 !! ng/μL
+
! Time (min) !! 0 !! 20 !! 40 !! 60 !! 80 !! 100 !! 120
|-
|-
-
| AgrA || 1.66 || 16.4
+
| OD650 || 0.1487 || 0.1541 || 0.1642 || 0.1980 || 0.2470 || 0.3205 || 0.3643
|-
|-
-
| AgrB || 1.91 || 23.5
+
|}
 +
 
 +
- t0 = 120min
 +
 
 +
- Follow the protocol of transformation
 +
 
 +
- Plasmid miniprep of ECE153, 166, 171 (to make the transformation)
 +
 
 +
- Nanodrop ( to add 0.5μg of DNA for transformation)
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
| AgrC || 1.99 || 35.9
+
!  Vetor !! 260/280 !! μg/mL !! quantity of DNA to add for transformation (μL)
|-
|-
-
| AgrD || 2.13 || 4.9
+
| ECE153 || 1.5 || 18.4 || 27.2
 +
|-
 +
| ECE166 || 1.83 || 39.5 || 12.7
 +
|-
 +
| ECE171 || 1.83 || 115.8 || 4.4
 +
|}
 +
 
 +
- Plate (with appropriate antibiotics)
 +
* ECE166 : Cm5
 +
* ECE153 : Spc50
 +
* ECE171 : Kan5 (to prepare agar plate, 40μL of Kan 25mg/mL into 200mL of agar)
 +
 
 +
==Check our stock of ECE171==
 +
 
 +
- We made big stocks of ECE171, before keeping it, we wanted to check it, so we run double digest on a gel!!
 +
 
 +
- Results : problem!!
 +
 
 +
- 2 possible causes : Double digest was made " days before and kept in the freezer (possible degradation) + too much DNA on the gel
 +
 
 +
- New double digest (from pellets in the freezer) + new gel
 +
 
 +
- Results : ok!
 +
 
 +
==Glycerol stocks==
 +
 
 +
- for IA751 and IA771, add 100μL of culture and 500μL of glycerol (60%)
 +
 
 +
==Xylose experiment==
 +
 
 +
- To test the transformation of ECE153, we want to induce the promoter Pxyl, and we will have green fluorescence
 +
 
 +
- Add 1mL of culture IA751 + ECE153 (from yesterday), 8mL of LB and 45μL of Spc50, 1mL of xylose (1g into 10mL)
 +
 
 +
==Plasmid miniprep for B.S.==
 +
 
 +
- We first tried to use the same protocol than for E.coli (Zyppy kit) for ECE166 transformed in IA751
 +
 
 +
- Nanodrop to see the result
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
!  Vetor !! 260/280 !! μg/mL
 +
|-
 +
| ECE166, colony 1 || 1.69 || 11.2
 +
|-
 +
| || 1.41|| 14.5
 +
|-
 +
| ECE166, colony 2|| 1.34 || 7.0
 +
|}
 +
 
 +
- We have very low concentration of DNA, we will digest that plasmid tomorrow morning to see if it is ECE166, and if it does work, we will try to modify our protocole tomorrow
 +
 
 +
==Transformation of ECE188, 189, 190 in E.coli==
 +
 
 +
* We received this vector in DNA stocks, so we have to transform them to test them 9because we do not have enough DNA)
 +
* Use TOP10 competent cells
 +
* Pellet, add 100μL of CaCl2 solution, for each transformation, use 50μL of cells
 +
* Add 2μL of DNA, and 1μL of PUC19 for control
 +
* 30min on ice
 +
* 2min at 42°C, 2min on ice
 +
* 2h at 37°C
 +
* Plate on Amp100 plates
 +
 
 +
==In Preparation of Beta-galactosidase Assay (Promoter Assay PA)==
 +
 
 +
Biobrick Extraction:
 +
 
 +
E0040
 +
* GFP (Amp resistance)
 +
* Extracted from 2007 Plate 1 Well 5H
 +
I13522
 +
* E.coli constitutive promoter with RBS and GFP (Amp resistance)
 +
* Extracted from 2007 Plate 3 Well 13C
 +
B0034
 +
* E. coli RBS (Amp resistance)
 +
* Extracted from 2008 Plate 1000 Well 2E
 +
R0040
 +
* E. coli promoter (Amp resistance)
 +
* Extracted from 2008 Plate 1000 Well 4C
 +
 
 +
Transformation of competent TOP10 with the 4 biobricks above along with pUC19 control
 +
* 20μL TOP10 used for each transformation with 2μL DNA
 +
* Followed standard protocol
 +
* Plated out neat and 1/10 on Amp100 plates after 2 hours of incubation, then plates are kept at 37°C overnight
 +
 
 +
=August 14th=
 +
 
 +
== Results of transformation==
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
| Pxyl || 1.54 || 5.6
+
!  Vector !! Antibiotic !! number of colonies !! Transformation efficiency
|-
|-
-
| Ppac || 1.49 || 4.6
+
| ECE166 || Cm5 || 31 || 62
|-
|-
-
| Pspc || 1.62 || 9.6
+
| Control || Cm5 || 0 ||
|-
|-
-
| Pupp || 1.88 || 8.5
+
| ECE171 || Kan5 || 5 + a lot of small colonies || ?
|-
|-
-
| RBS S || 2.44 || 29
+
| Control || Kan5 || 1 + 5 very small ||
|-
|-
-
| RBS W || 1.44 || 10.7
+
| ECE153 || Spc50 || about 25 (on a side) || 50
|-
|-
 +
| Control || Spc50 || 6 + 2 very small  ||
|}
|}
-
- Extract plasmid annd Agr from gel and clean
+
- Problem with our control, maybe we should check the resistance of competent cells (before transformation)
-
- Ligation
+
==BioBrick extraction for testing promoters and RBS in ''B.subtillis''==
 +
The primers for inducible ''B.subtillis'' promoters have been ordered. Meanwhile, we would like to be able to compare the RBS and promoter strengths in ''E.coli'' and ''B.subtillis'', using GFP fluorescence to quantify gene expression.
 +
We attempted to isolate 4 BioBricks: I13522 (GFP under constitutive promoter), E0040 (GFP only), R0040 (a promoter), & B0034 (an ''E.coli'' RBS).
 +
So far, only the former two, extracted from 2007 wells, grew after transformation. Single-colony PCR was used to test the transformants.
-
=August 21st=
+
Expected VF2-VR fragment sizes:
-
==Transformation of ligation products==
+
I13522 -  ~2.4 kb
 +
E0040 -  958 bp
 +
R0040 -  292 bp
 +
B0034 -  250 bp
-
- Spin chemically competent TOP10, add 100μL of CaCl2 solution
+
==Transformation of vectors 188, 189, 190==
-
- Add 5μL of DNA (ligation products), and 1μL of PUC9
+
- Transformation of yesterday did not work! there was nothing on plates, even on the control plate
-
- Continue the protocol of transformation
+
- Try again, same protocol, 2μL of DNA, control : PUC9
-
==J04630==
+
- Plate on Amp100 for control and Amp100 + Cm5 for vectors
-
- Plate good colonies from yesterday on Kan25 and put in LB (for plasmid stock for tomorrow)
+
==Double digest of ECE166 (extracted from transformed Bacillus yesterday)==
-
==Plasmid stocks==
+
- Transformation from 07/08
-
- Plasmid miniprep R0040 and R0062
+
- 16μL of DNA, 2μL of Buffer EcoRI, 1μL of EcoRI (Biolab) and 1μL of HindIII
 +
- Incubator at 37°C for 35min, heat shock at 80°C for 5min
-
=August 23rd=
+
- Run on a gel with 4μL of DNA
-
==Check promoters (after PCR from bacillus vectors)==
+
* Result : no bands! not enough DNA!
-
- Gel
+
- Run on a new gel with 16μL of DNA
-
* Lane1 : Hyperladder 5
+
* Result : " bands, one of about 2000b, and one of about 5000b> The big one seem to be too small...
-
* Lane2 : Pxyl
+
We are going to check this transformation with fluorescence too!
-
* Lane3 : Pspac
+
-
* lane4 : Ppac
+
-
* Lane5 : Pupp
+
-
[[Image:photoeds.gif|250px|center]]
+
==Erythromycin plate==
-
Size is ok.
+
- Erythromycin plate for the transformation 13/08/08
 +
- IA771 (control, they should survive)
-
=August 27th=
+
- IA771 + ECE166 (non integration vector, so colonies should survive)
-
==New transformation of ligation products==
+
- IA771 + ECE171 (integration vector but in another locus, should survive)
-
* Products : agrD, Pupp, Pspac, RBS W, RBS S (from ligation with biolabs kit)
+
-
- Competent top 10 from the freezer
+
- IA771 + 153 9integration vector, they should die if they are transformed)
-
- Add 5μL of ligation products, and 1.2μL of PUC9
+
==New stocks==
 +
- LB stocks with antibiotic of ECE151, 153, 166
-
=August 28th=
+
- LB stock of ECE153 with Spc50 (one from colonies from LB stock from 11/08 and one from 13/08 transformation plates)
-
==Results from transformation of ligation products==
+
==Check fluorescence with microscope==
-
- nothing on plate, even on control plate
+
* ECE166 from the plate from 13/08
-
*Reasons ?
+
We diluted one colony from this plate into SDW and observed with microscope.
-
- cells non competent anymore (try with fresh competent cells)
+
Result : Bacillus is fluorescent! There are some bacteria which are brighter than others, but it is because it is not an integration vector. This transformation worked!!!
-
- not enough DNA (try with 10μL of DNA)
+
* ECE153
-
- very short ligation products
+
We added xylose, but we did not incubate our sample after that, so it did not work> We will try again tomorrow with a period of incubation> 
-
==Transformation of ligation products (new)==
+
We will have to
-
* Products to ligate : Pupp, Pspac, agrD, RBS S and RBS W
+
==Plates from yesterday for Biobrick Extraction (PA)==
-
- new fresh competent TOP 10
+
* Growth observed on Amp100 plates for I13522 and E0040 only but not for R0040, B0034 and pUC9 control
 +
* Re-plated overnight culture neat on Amp 75 plates and incubate at 37°C overnight
-
- 5μL of DNA (1.5μL of PUC9)
+
==== Single Colony PCR of I13522 and E0040====
-
- 2h30 in the incubator
+
* 5 colonies picked from each neat agar plate onto Amp75 plates
 +
* PCR following standard protocols
 +
* Run PCR product on 1.2% agarose gel at 70V which is later soaked in EtBr
 +
First Row:
 +
* Lane 2 - Hyperladder IV
 +
* Lane 3-7 - I13522 Colonies 1-5 PCR
 +
* Lane 9-13 - E0040 Colonies 1-5 PCR
 +
Second Row:
 +
* Lane 2 - Hyperladder IV
 +
* Lane 3-6, 9 - R0010 Colonies 1-5 PCR
-
=August 29th=
+
Result:
 +
* Expected size of band: I13522 - 2375bp; E0040 - 958bp; R0010 - 292bp
 +
* Picked colonies 4 and 5 for E0040 into 10ml LB with Amp100 and incubate overnight at 37°C as the band corresponds to about 958bp
 +
* Band of R0010 colonies 3 and 5 correspond to 292bp and will incubate in LB tomorrow when colonies have grown on agar plates
-
==Results of transformation with our ligation products==
+
=August 15th=
-
* Everything grew! Better efficiency with electrop. than with chemical protocol
+
==Transformation ECE188, 189, 190 into E.coli==
-
==Single colony PCR to check our transformation==
+
- Results : only our control plate grew!!
 +
- Maybe we did not add enough DNA, or there is another problem. We will e mail the guy from Bacillus center before trying again, because we don't have enough DNA.
 +
==Plasmid miniprep ECE 151 (x2), ECE 153, ECE 166==
 +
 +
*'''Nanodrop results''' :
{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
!   Transformed products !! number of picked colonies from chemical transformation !! number of picked colonies from electrop. transformation (neat) !! number of picked colonies from electrop. transformation (1:10)
+
! !! 260/280 !! ng/μL
|-
|-
-
| Pupp || 2 || 2 || 0
+
| ECE 151 (3) || 1.79 || 86.8
|-
|-
-
| Pspac || 2 || 2 || 0
+
| ECE 151 || 1.88 || 106.3
|-
|-
-
| RBS S || 3 || 0 || 2  
+
| ECE 153 || 1.80 || 32.2
|-
|-
-
| RBS W || 3 || 0 || 2
+
| ECE 166 || 1.87 || 36.2
|-
|-
-
| agrD || 3 || 2 || 0
 
|}
|}
-
- Single colony PCR : 13μL of SDW+cells, 5μL of Master Mix, 1μL of VF and 1μL of VR (and plate each single colony)
 
-
- Load a gel (1.3% agarose) : 5μL of PCR products + 1μL of dye (only 1μL of 100b ladder)
+
==Xylose Induction with ECE 153==
-
In the death plasmid, the VF-VR size is about 280b.
+
- 4 Tubes
 +
 
 +
- No fluorescence! We have to think about the transformation of integration vectors into Bacillus.
 +
 
 +
 
 +
==Glycerol Stock Transformation==
 +
 
 +
- Spin down cells (competent) from 5/8 and 13/8 stocks
 +
 
 +
- Remove liquid, resuspend in Medium B
 +
 
 +
- Incubate for 60 mins at 37°C
 +
 
 +
- Add ECE 166 into tubes
 +
 
 +
- Incubate for 30 mins at 37°C
 +
 
 +
- Plate out on CM 5 plates
 +
 
 +
- 4 plates
 +
 
 +
* 771 (5/8) + ECE 166
 +
 
 +
* 771 (5/8) Control
 +
 
 +
* 771 (13/8) + ECE 166
 +
 
 +
* 771 (13/8) Control
 +
 
 +
 
 +
 
 +
==Transforming ECE 188, 189, 190 (3rd try)==
 +
 
 +
- 2 tubes of chemically competent top 10
 +
 
 +
- Spin down, remove liquid
 +
 
 +
- Resuspend cells in 100μL of CaCl2 50mM
 +
 
 +
- 4 tubes with 50μL of cells
 +
 
 +
* ECE 190 (all)
 +
 
 +
* ECE 189 (all)
 +
 
 +
* ECE 188 (all)
 +
 
 +
* PUC 9 (5μL) [ Control ]
 +
 
 +
- Ice for 30 mins
 +
 
 +
- 42°C for 2 mins
 +
 
 +
- Ice for 2 mins
 +
 
 +
- Incubate at 37°C for 2 hours
 +
 
 +
- Plate out on Amp 100 (Neat)
 +
 
 +
==Biobrick E0040 and I13522 (PA)==
 +
 
 +
* PCR E0040 colonies 4 and 5 after miniprep of plasmid to ensure plasmid identity again
 +
* Protocol:
 +
**SDW 7.5μL
 +
**MasterMix 10μL
 +
**VR and VF2 1x2μL
 +
**DNA sample 0.5μL
 +
* Run on 1.2% SyBr E-gel
 +
* Lane 2 - 100bp ladder
 +
* Lane 3 - E0040 colony 4
 +
* Lane 4 - E0040 colony 5
 +
* Result: Bands at around 1000bp on both lanes 3 and 4 with a brighter band for lane 3.  Correct band as E0040 VF-VR is 958bp
 +
* Minprep of plasmid E0040 from colony 4 is kept
 +
 
 +
* Transform chemically compotent TOP10 with I13522 biobrick extract again
 +
* Transformation following standard protocols
 +
* TOP10 with I13522 plated neat and 1/10 on Amp. 100 plates and incubate at 37°C overnight
 +
* Will pick single colonies tomorrow for PCR analysis and into LB with Amp100
 +
 
 +
=August 16th=
 +
 
 +
==Result from the days before==
 +
*For Ery plates
 +
 
 +
- Everything grew, we will try again to see if there is a problem with the antibiotic (and we will do a negative control with the strain IA751)
 +
 
 +
* Transformation of E.coli with ECE188, 189, 190
 +
 
 +
- We have a result from our 3 transformation in E.coli. To transform E.coli with these vectors we have to wait for 2 days before seeing cells!! We will check the vectors next week.
 +
 
 +
* Transformation of B.S. IA771 (glycerol stocks)
 +
 
 +
- Nothing! Problem with the storage of competent IA771???
 +
 
 +
=August 18th=
 +
 
 +
==Test Ery efficiency==
 +
 
 +
Since all Ery plates grew, we want to check the antibiotic stock.
 +
 
 +
- New Ery plate of IA751 (they should die)
 +
 
 +
- New LB stock (Ery5) with IA751
 +
 
 +
==Single colony plates==
 +
 
 +
- New plates (Cm5 + Amp100) with single colony of ECE188 (from 14/08), ECE189 (from 13/08) and ECE190 (from 14/08)
 +
 
 +
- Grow the same single colony into LB with antibiotic
 +
 
 +
==Transformation of glycerol stocks of B.S.==
 +
 
 +
The result from the last transformation of glycerol stocks was negative> We tried again with two different protocols.
 +
 
 +
* '''First protocol'''
 +
 
 +
- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B
 +
 
 +
- Incubate about 1h15
 +
 
 +
- Add 0.6μg of ECE166 (because we managed to transform BS with this vector)
 +
 
 +
- Incubate 1h30
 +
 
 +
- Plate on Cm5 plates (DNA less control and transformed cells)
 +
 
 +
* '''Second protocol'''
 +
 
 +
- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B
 +
 
 +
- Add 0.6μg of ECE166 (because we managed to transform BS with this vector)
 +
 
 +
- Incubate 30min
 +
 
 +
- Plate on Cm5 plates (transformed cells)
 +
 
 +
 
 +
- Plate cells after 10min of incubation (without DNA) on blank plates (to see if they are alive)
 +
 
 +
== PA with I13522, E0040 and B0034 Preparation==
 +
 
 +
* R0040 TOP10 miniprep from LB culture done by Kevin
 +
* 5 single colonies of I13522 picked onto Amp100 plate and LB with Amp100 done on Sunday
 +
* Miniprep of the 5 colonies with sinigle colony PCR
 +
* PCR followed standard protocol and product is run on 0.8% EtBr E-gel
 +
* iGEM34 porgramme is used for PCR
 +
Gel:
 +
* 4μL dye with 20μL PCR product
 +
* 5μL Hyperladder I with 15μL SDW into lane 2
 +
* Lanes 3-7: I13522 Colonies 1-5
 +
* Lane 8: R0040
 +
* Lane 9: Hyperladder I
 +
Result:
 +
* Size of I13522 is not right as the bands are all around 4500bp but it should be 2375bp
 +
* R0040 is of the correct size
 +
 
 +
* Transformed chemically compotent TOP10 with B0034 biobrick extract and pUC9 control
 +
* Plate on Amp100 plates after standard transformation
 +
 
 +
=August 19th=
 +
 
 +
==Results from the transformation with glycerol stocks==
 +
 
 +
We used two different glycerol stocks, one from 07/08 and one from 13/08. We also had 2 different protocols (cf 18/08 page).
 +
 
 +
We tested our stocks on blank plates, cells are alive, the problem is to know if they are still competent or not.
 +
 
 +
* '''Protocol 1 (with incubation time before adding DNA)'''
 +
 
 +
- Stock from 07/08 : nothing on the control and nothing on the transformation plate.
 +
 
 +
- Stock from 13/08 : nothing on the control and nothing on the transformation plate.
 +
 
 +
* '''Protocol 2 (without incubation time before adding DNA)'''
 +
 
 +
- Stock from 07/08 : nothing on the control and nothing on the transformation plate.
 +
 
 +
- Stock from 13/08 : nothing on the control, and 1 colony on the transformed plate. We checked the fluorescence of bacteria into this colony (vector ECE166 with promoter and GFP). We have fluorescence, so our transformation is ok.
 +
 
 +
The result of this confirm that our stock of competent cells in glycerol is still competnet since we managed to transform one colony. The problem is certainly not the storage, but our protocol when we use frozen competent cells> We need to improve the efficiency of this protocol.
 +
 
 +
==Test our Erythromycin stock==
 +
 
 +
- We put some colonies on a Ery 0.5 plate. They grow, but in a very small amount, so our antibiotic seems to be fine.
 +
 
 +
- Cells in LB with antibiotic : no growth!
 +
 
 +
So our antibiotic is ok, either we did not transform our cells and that is why everything grew on Ery plates, either there is a problem in our protocol for Ery test.
 +
 
 +
==Plasmid miniprep of ECE153, ECE166 and ECE171==
 +
 
 +
- Plasmid miniprep LB stocks from yesterday
 +
 
 +
- Nanodrop
{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
!   Transformed products !! size of the product (with cutting sites) !! expected size after PCR (about)
+
! !! 260/280 !! ng/μL
|-
|-
-
| Pupp || 255 || 480
+
| ECE 153 || 1.63 || 32.6
|-
|-
-
| Pspac || 125 || 350
+
| ECE 166 (1) || 1.73 || 56.2
|-
|-
-
| RBS S || 56 || 280
+
| ECE 166 (2) || 1.67 || 57.7
|-
|-
-
| RBS W || 56 || 280
+
| ECE 171 || 1.81 || 185.5
|-
|-
-
| agrD || 200 || 430
 
|}
|}
-
* Result : nothing, even no ladder, problem with the gel!
+
==Transformation of IA771==
-
- run again on a e-gel
+
We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171.
-
* Lane1 :ladder 100bp
+
Spectrophotometer : blank made with medium A
-
* Lane2 : Pupp colony 1
+
-
* Lane3 : Pupp colony 3
+
-
* Lane4 : Pspac colony 1
+
-
* Lane5 : Pspac colony 3
+
-
* Lane6 : RBS S colony 1
+
-
* Lane7 : RBS S colony 4
+
-
* Lane8 : RBS W colony 1
+
-
* Lane9 : RBS W colony 4
+
-
* Lane10 : agrD colony 1
+
-
* Lane11 : agrD colony 4
+
-
* Lane12 : HyperladderI
+
-
[[Image:photor.gif|300px|center]]
+
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
! Time (min) !! 0 !! 20 !! 40 !! 80
 +
|-
 +
| OD650 || 0.1512 || 0.1498 || 0.1476 || 0.1535
 +
|-
 +
|}
-
*Result : nothing, just the primers! Problem with our PCR
+
There was a problem with our colonies. At t=80min, we inoculated again our tube of Medium A.
-
==Plate biobricks from MIT==
+
- We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171
-
*E0840
+
- Spectrophotometer : blank made with medium A
-
*B0014
+
-
*I712007
+
-
*C0012
+
-
*B0015
+
-
*C0061
+
-
*R0063
+
 +
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
! Time (min) !! 0 !! 20 !! 40 !! 60 !! 80 !! 100
 +
|-
 +
| OD650 || 0.2393 || 0.2504 || 0.2663 || 0.2807 || 0.3184 || 0.3672
 +
|-
 +
|}
-
=September 2nd=
+
[[Image:Book1_3816_image001.gif|500px|center]]
-
==Check transformation in E.coli of our ligated products==
+
- Continue the protocol, add DNA (ECE153, 166, 171)
-
- Single colony PCR with VF and VR : add 10μL of SDW, 5μL of MM, 1μL of VF, 1μL of VR and 3μL of cells
+
- Plate on antibiotic plates
 +
 
 +
* New glycerol stocks
 +
 
 +
- 8 tubes : spin cells, throw away the supernatant, add 500μL of glycerol 60%
 +
 
 +
- 8 tubes : spin cells, keep only 100μL of medium B, resuspend cells and add 500μL of glycerol 60%
 +
 
 +
- Put them in the freezer at -80°C
 +
 
 +
==Dealing with R0040, R0062, S0168, I13522 for PA==
 +
 
 +
* Plated R0040, R0062, S0168 received from MIT onto Amp100 plates and incubate at 37°C
 +
* Pciked 4 single colonies for each after colonies have grown and incubate overnight
 +
 
 +
Check I13522 biobrick DNA
 +
* PCR biobrick DNA from 2007 wells
 +
* Run 0.8% EtBr E-gel
 +
* Lane 5: I13522
 +
* Lane 7: Hyperladder I
 +
Result:
 +
* Size of band is too small under 1000bp should be 2375bp instead
 +
* Transform TOP10 again but with I13522 from 2008 wells
 +
 
 +
=August 20th=
 +
 
 +
==Check vector ECE190==
 +
 
 +
- Plasmid miniprep
 +
 
 +
- Nanodrop
 +
 
 +
- Double digest : 6μL of SDW, 8μL of DNA, 1μL of buffer, 1μL of XbaI and 1μL of PstI
 +
 
 +
- Run on a gel
 +
 
 +
[[Image:Boodfg_image001.gif|50px|center]]
 +
 
 +
- '''Result''' : 2 bands (lane3, a little bit more than 3000b and a little bit more than 5000b) :ok!
 +
 
 +
=August 21st=
 +
 
 +
==Check ECE188==
 +
 
 +
- Plasmid miniprep
 +
 
 +
- Double digest with XbaI and PstI
 +
 
 +
- Gel (lane6)
 +
 
 +
[[Image:photop.gif|200px|center]]
 +
 
 +
- Result : nothing! not enough DNA
 +
 
 +
=August 22nd=
 +
 
 +
==Stock ECE112, 166, 153==
 +
 
 +
- Plasmid miniprep (2 tubes of ECE112, 2 tubes of ECE166 and 1 tube of ECE153)
 +
 
 +
- Nanodrop
 +
 
 +
==Transformation of Bacillus==
 +
 
 +
* For strain IA751 : transform with ECE166, 171, 153 and 112
 +
 
 +
* For strain IA771 : transform with ECE166, 171
 +
 
 +
* for glycerol stocks (one with 100μL of medium B and glycerol, and one with only glycerol) : transform with ECE166
 +
 
 +
 
 +
- We used the same protocol than before with a few changes :
 +
 
 +
* add sterile 50% glucose in medium A
 +
* don't keep samples after using them for spectrophotometer
 +
* spin each tubes and keep only 100μL of medium A (to concentrate cells) before adding DNA
{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
! inserted products !! number of colonies to check (chemical transf.) !! number of colonies to check (electrop. transf.)
+
! Time (min) !! 0 !! 20 !! 40 !! 60 !! 80 !! 100 !! 120 !! 130 !! 140
 +
|-
 +
| OD650 for IA751 || 0.1817 || 0.1870 || 0.1929 || 0.2115 || 0.2543 || 0.3291 || 0.3695 || 0.4188 ||0.4224
 +
|-
 +
| OD650 for IA751 ||0.1818 || 0.1874 || 0.1907 || 0.2088 || 0.2452 || 0.3182 || 0.3575 || 0.4228 || 0.4314
 +
|-
 +
|}
 +
 
 +
[[Image:graph.gif|500px|center]]
 +
 
 +
- For glycerol stocks, resuspend cells into 100μL of medium B, add DNA and wait for 30min, end plate them
 +
 
 +
=August 23rd=
 +
 
 +
==Results of transformation from 23/08/2008==
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
!  Vector !! Strain !! Antibiotic !! number of colonies !! Conclusion
 +
|-
 +
| ECE166 || IA771, glycerol stock with medium B || Cm5 || one large colony and a lot of very small colonies || Contamination?
 +
|-
 +
| Control || IA771, glycerol stock with medium B || Cm5 || a lot of very small colonies || Contamination?
 +
|-
 +
| ECE166 || IA771, glycerol stock without medium B || Cm5 || many very small colonies || Contamination?
 +
|-
 +
| Control || IA771, glycerol stock without medium B || Cm5 || a few tiny colonies || Contamination?
 +
|-
 +
| ECE166 || IA751, fresh || Cm5 || 9 large colonies, no small ones || to test
 +
|-
 +
| Control || IA751, fresh || Cm5 || no growth, scratches || ok
 +
|-
 +
| ECE166 || IA771, fresh || Cm5 || 1 large colony, many small worm-like growth, scratches? || to test
 +
|-
 +
| Control || IA771, fresh || Cm5 || no colonies at all || ok
 +
|-
 +
| ECE171 || IA771, fresh || Kan5 || no colonies || ??
 +
|-
 +
| Control || IA771, fresh || Cm5 || no colonies  || ok
|-
|-
-
| RBS S || 3 || 2
+
| ECE171 || IA751, fresh || Kan5 || about 30 large colonies and 100 small colonies || to test?
|-
|-
-
| RBS W || 3 || 2
+
| Control || IA751, fresh || Cm5 || 3 large colonies, about 20 small to medium colonies  || contamination?
|-
|-
-
| Pspac || 2 || 2
+
| ECE112 || IA751, fresh || Cm5 + Spc100 || no growth || ??
|-
|-
-
| Pupp || 2 || 2
+
| Control || IA751, fresh || Cm5 + Spc100 || no growth ||
|-
|-
-
| agrD || 3 || 2
+
| ECE153 || IA751, fresh || Spc50 || about 500 medium to large colonies || to test
|-
|-
 +
| Control || IA751, fresh || Spc50 || no growth || ok
|}
|}
-
- Gel (3% of agarose)
+
=August 26th=
-
[[Image:gel1.gif|200px|center]]
+
==Xylose test==
 +
* for ECE153
-
* Result
+
- Prepare 4 tubes with 10mL LB and Spc50, inoculate each tube with a colony from IA751 + ECE153
 +
 
 +
==Starch plate==
 +
 
 +
- prepare some blank agar plates
 +
 
 +
- add 1g of starch into 100mL of soft agar, mix, boil
 +
 
 +
- put a thin layer of soft agar with starch
 +
 
 +
- plate with IA751, IA771, IA751+ECE153 and IA751 (control plate, to see if we mixed some plates)
 +
 
 +
- Incubate at 37°C
 +
 
 +
==Ery plate==
 +
 
 +
- Plate IA751, IA771, IA751 (control plate) and IA751+ECE153
 +
 
 +
 
 +
 
 +
=August 27th=
 +
 
 +
==Xylose test==
 +
 
 +
* Preparation of xylose tube
 +
 
 +
- Add 1mL of culture from yesterday, 1mL of xylose (0.1g/mL) and 8mL of LB and 45μL of Spc (do that for the 4 different colonies)
 +
 
 +
- Incubate 5hours
 +
 
 +
- Observation with microscope : some green task, but they do not really match with bacillus cells, not conclusive!
 +
 
 +
==Starch plate==
 +
 
 +
- Add 5mL of iodine solution on starch plate, wait for 1min, throw the liquid
 +
 
 +
- Result
{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
! gel !! lane !! inserted product (name of the colony) !! observation !! conclusion
+
! Strain !! added vector !! Observation !! Conclusion
|-
|-
-
| top || 3 || RBS S (1) || one band (260bp) || size of the insert, RBS too small to see on a gel
+
| IA771 || none || no clearance zones || ok
|-
|-
-
| top || 4 || RBS S (2) || one band (260bp) || size of the insert, RBS too small to see on a gel
+
| IA751 || none || big clearance zones || ok
|-
|-
-
| top || 5 || RBS S (4) || nothing || ?
+
| IA751 || ECE153 || no clearance zones || trnsformation worked!
|-
|-
-
| top || 6 || RBS S (5) || nothing || ?
+
| IA751 or IA771 || none or ECE171 || big clearance zones || we have some colonues on control plate with IA751, contamination? or antibiotic resistance?
|-
|-
-
| top || 7 || RBS W (1) || 2 band (260bp + 350bp) || size of the insert, RBS too small to see on a gel + something else?
+
|}
 +
 
 +
==Ery test==
 +
 
 +
- IA771 alive and IA751 dead, confirmation that we did not mix plates (same conclusion than with starch plate, last line)
 +
 
 +
==Test glycerol stocks of competent bacillus==
 +
 
 +
- Pellet bacillus cells, add 100μL of prewarmed mediumB, add 0.6μg of DNA, incubate 30min at 37°C by shaking, plate
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
| top || 8 || RBS W (2) || one band (350bp) || too big
+
!  Strain !! added vector !! Antibiotic
|-
|-
-
| top || 9 || RBS W (4) || one band (260bp) || size of the insert, RBS too small to see on a gel
+
| IA771 || none || blank plate (to check they are alive)
|-
|-
-
| top || 10 || RBS W (5) || one band (260bp) || size of the insert, RBS too small to see on a gel
+
| IA771 || none || Cm5 (negative control)
|-
|-
-
| top || 11 || Pspac (1) || 2 band (260bp + 350bp) || maybe problem with products loaded on gel (exactly the same bands than for RBS W)
+
| IA771 || ECE166 || Cm5
|-
|-
-
| top || 12 || Pspac (2) || one band (450bp) || size of Pupp?
+
| IA751 || none || blank plate (to check they are alive)
|-
|-
-
| top || 13 || Pspac (3) || one band (450bp) || size of Pupp?
+
| IA751 || none || Cm5 (negative control)
|-
|-
-
| bottom || 3 || Pspac (4) || nothing || ?
+
| IA751 || none || Spc50 (negative control)
|-
|-
-
| bottom || 4 || Pupp (1) || one band (400bp) || size of Pspac?
+
| IA751 || ECE112 || Cm5
|-
|-
-
| bottom || 5 || Pupp (2) || one band (400bp) || size of Pspac?
+
| IA751 || ECE153 || Spc50
 +
|}
 +
 
 +
=August 28th=
 +
 
 +
==Results from Bacillus transformation with glycerol stocks==
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
| bottom || 6 || Pupp (3) || one band (slightly lower than 400bp) || size of Pspac?
+
!  Strain !! added vector !! Antibiotic !! number of colonies !! Conclusion
|-
|-
-
| bottom || 7 || Pupp (4) || one band (400bp) || size of Pspac?
+
| IA771 || none || blank plate || confluent colonies || cells are alive
|-
|-
-
| bottom || 8 || agrD (1) || 2 bands (380 and 450bp) || big band is ok
+
| IA771 || none || Cm5 || 0 || no contamination
|-
|-
-
| bottom || 9 || agrD (2) || 2 bands (380 and 450bp) || big band is ok
+
| IA771 || ECE166 || Cm5 || 2 || transformation ok, very low efficiency
|-
|-
-
| bottom || 10 || agrD (3) || no bands || ?
+
| IA751 || none || blank plate || confluent colonies || cells are alive
|-
|-
-
| bottom || 11 ||agrD (4) || strong band (450bp) || ok
+
| IA751 || none || Cm5 || 0 || no contamination
|-
|-
-
| bottom || 12 || P2 || one band (350bp) || ok
+
| IA751 || none || Spc50 || 0 || no contamination
|-
|-
 +
| IA751 || ECE112 || Cm5 || 0 || problem, we can not manage to transform this vector
 +
|-
 +
| IA751 || ECE153 || Spc50 || 156 || transformation ok, good efficiency
|}
|}
-
=September 3rd=
+
==Test AmyE insertion==
-
==Checking the insert of promoters==
+
* Colonies PCR for Bacillus (IA751 transformed with ECE153, IA751, and IA771)
-
We ligated promoters into death vector, and transforned into Top 10. Our transformation worked, but it was hard to say with the result of yesterday if we had the good insert. Moreover, it is possible that we inverted Pupp and Pspac. So we are going to make a new single colony PCR with the primers of promoters (we used the same single colonies than yesterday).
+
- 20μL of lysozyme, a few colonies for each
-
We also want to PCR again all promoters (Pspac, Pxyl, Pupp and Ppac) from plasmids to have more.
+
- Cycle : 15min qt 37°C, 15min at 99°C, 1min at 4°C, 1min at 99°C and 1min at 4°C
-
Picture of the gel with the different promoters
+
- for each PCR : 12μL of cells, 1μL of cells, 1μL of primer1, 1μL of primer2, 5μL of master mix
-
[[Image:gel5.gif|300px|center]]
+
- PCR (iGEM program)
 +
- run a gel
 +
 +
[[Image:photoq.gif|150px|center]]
 +
 +
* Result : nothing on the gel!!! But we can not manage to PCR anything... there is a problem in our PCR protocol, or products!
 +
 +
=August 29th=
 +
 +
==Stock of ECE112==
 +
 +
- Bulk up ECE112 (Amp100) rfom 10mL LB (28/08), take 1mL and add to 100mL of LB in big flask
 +
 +
- Grow overnight in 37°C incubator with vigorous shaking
 +
 +
- Aliquot in 10mL tubes, pellet and freeze cell pellets
 +
 +
==Transformation of glycerol stock==
 +
 +
* Strian : IA751
 +
 +
- Transfer into Eppendorf tubes and spin down for 30min
 +
 +
-Remove glycerol and add medium B
 +
 +
- Add ECE112 and ECE153, and one control tube
 +
 +
- incubate for 30min in 37°C
 +
 +
- Plate out ECE112 (Cm5), ECE153 (Spc50), DNA less (blank, Cm5, Spc50)
 +
 +
=September 1st=
 +
 +
==Result for Bacillus transformation (27/08)==
{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
! promoter !! colony !! primers !! name
+
!   Strain !! Vector !! Antibiotic !! number of colonies !! Conclusion
|-
|-
-
| Pspac || 1 || Pspac primers || A
+
| IA751 || None || Spc50 || 0 || ok
|-
|-
-
| Pspac || 1 || Pupp primers || B
+
| IA751 || ECE153 || Spc50 || about 200 || very good efficiency
|-
|-
-
| Pspac || 2 || Pspac primers || C
+
| IA751 || None || Blank || confluent || cells are alive
|-
|-
-
| Pspac || 2 || Pupp primers || D
+
| IA751 || None || Cm5 || a lot || problem of contamination (bacillus??? or E.coli???)
|-
|-
-
| Pupp || 1 || Pupp primers || E
+
| IA751 || ECE112 || Cm5 || a lot || to check
 +
|}
 +
 
 +
==New Bacillus transformation (to make glycerol stocks)==
 +
 
 +
*2 attempts with plates from 12/08 and 18/08, no growth... Cells may be dead.
 +
 
 +
*New plate of IA771 and IA751 (with glycerol stocks)
 +
 
 +
=September 2nd=
 +
 
 +
==Transformation of Bacillus==
 +
* with fresh plates from yesterday, last protocol
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
| Pupp || 1 || Pspac primers || F
+
! Time (min) !! 0 !! 20 !! 40 !! 60 !! 85 !! 105 !! 125
|-
|-
-
| Pupp || 3 || Pupp primers || G
+
| OD650 for IA751 || 0.1192 || 0.1330 || 0.1736 || 0.2238 || 0.3124 || 0.4523 || 0.5113
 +
|-
 +
| OD650 for IA771 ||0.1144 || 0.1216 || 0.1593 || 0.2296  || 0.3476 || 0.4302 || 0.5565
|-
|-
-
| Pupp || 3 || Pspac primers || H
 
|}
|}
-
- Single colony PCR : add 10μL of SDW, 5μL of MM, 1μL + 1μL of primers, 3μL of cells
+
[[Image:transf0209.gif|500px|center]]
-
- Gel
+
*t0 (IA751) = 100min
-
* lane 3 : hyperladderI
+
*t0 (IA771) = 110min
-
* lane 4 : Pspac (colony1) with Pspac primers
+
-
* lane 5 : Pspac (colony1) with Pupp primers
+
-
* lane 6 : Pspac (colony2) with Pspac primers
+
-
* lane 7 : Pspac (colony2) with Pupp primers
+
-
* lane 8 : Pupp (colony1) with Pupp primers
+
-
* lane 9 : Pupp (colony1) with Pspac primers
+
-
* lane 10 : Pupp (colony3) with Pupp primers
+
-
* lane 11 : Pupp (colony3) with Pspac primers
+
-
* lane 12 : HyperladderI
+
-
[[Image:gel4.gif|300px|center]]
+
*Transformation
-
- Results
+
- IA751 on Cm5 and Spc 50 (control DNA less plates)
 +
 
 +
- IA751 + ECE112 (1μg of DNA) on Cm5
 +
 
 +
- IA751 + ECE153 (0.6μg of DNA) on Scp50
 +
 
 +
- IA771 on Cm5 (control)
 +
 
 +
- IA771 + ECE166 on Cm5
 +
 
 +
* Glycerol stocks (about 20 tubes of each)
 +
 
 +
==Check ECE112 stock==
 +
 
 +
- Double digest with XbaI and EcoRI
 +
 
 +
[[Image:gel2.gif|200px|center]]
 +
 
 +
- Gel : problem only one band (single digest???)
 +
 
 +
=September 3rd=
 +
 
 +
==Result from bacillus transformation with fresh competent cells (02/09)==
{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
! promoter !! colony !! primers !! observation !! Conclusion
+
! strain !! antibiotic !! added vector !! observation !! conclusion
|-
|-
-
| Pspac || 1 || Pspac primers || nothing || it is in fact Pupp
+
| IA751 || none || none || confluent colonies || cells are alive
 +
|-
 +
| IA751 || Spc50 || none || no colonies || no contamination
 +
|-
 +
| IA751 || Spc50 || ECE153 || 70 colonies || transformation to check, efficiency : about 115 colonies/μg of DNA
 +
|-
 +
| IA771 || none || none || confluent colonies || cells are alive
 +
|-
 +
| IA771 || Cm5 || none || no colonies || no contamination
 +
|-
 +
| IA771 || Cm5 || ECE166 || 16 colonies || transformation to check, efficiency : about 27 colonies/μg of DNA
 +
|-
 +
| IA771 || Cm5 || ECE112 || 41 small colonies || transformation to check, efficiency : about 41 colonies/μg of DNA
 +
|}
 +
 
 +
With 1μg of DNA, we manage to obtain colonies with ECE112. We will check our transformation with starch plates.
 +
 
 +
==Check ECE112 stock==
 +
 
 +
- Single and double digest with XbaI and EcoRI
 +
 
 +
[[Image:gel3.gif|300px|center]]
 +
 
 +
- Gel
 +
* Lane 3 : HyperladderI
 +
* Lane 4 : ECE112, 100mL flask, single digest with XbaI (expected size about 10,000bp)
 +
* Lane 5 : ECE112, 100mL flask, single digest with EcoRI (expected size about 10,000bp)
 +
* Lane 6 : ECE112, 100mL flask, double digest (expected size about 3200bp and 6,900bp)
 +
* Lane 7 : ECE112, 10mL flask, single digest with XbaI (expected size about 10,000bp)
 +
* Lane 8 : ECE112, 10mL flask, single digest with EcoRI (expected size about 10,000bp)
 +
* Lane 9 : ECE112, 10mL flask, double digest (expected size about 3200bp and 6,900bp)
 +
* Lane 10 : HyperladderI
 +
 
 +
- Results : ok
 +
 
 +
==Test glycerol stocks (from 02/09) of competent bacillus==
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
|-
|-
-
| Pspac || 1 || Pupp primers || band (about 280bp) || Pupp OK
+
! strain !! antibiotic !! added vector !! quantity of DNA
|-
|-
-
| Pspac || 2 || Pspac primers || nothing || it is in fact Pupp
+
| IA751 || none || none || 0
|-
|-
-
| Pspac || 2 || Pupp primers || band (about 280bp) || Pupp OK
+
| IA751 || Spc50 || none || 0
|-
|-
-
| Pupp || 1 || Pupp primers || nothing || it is in fact Pspac
+
| IA751 || Spc50 || ECE153 || 0.6
|-
|-
-
| Pupp || 1 || Pspac primers || band (about 180bp) || Pspac OK
+
| IA771 || none || none || 0
|-
|-
-
| Pupp || 3 || Pupp primers || nothing || it is in fact Pspac
+
| IA771 || Cm5 || none || 0
|-
|-
-
| Pupp || 3 || Pspac primers || band (about 180bp) || Pspac OK
+
| IA771 || Cm5 || ECE166 || 0.6
|}
|}
-
==Make some stock of our new biobricks==
+
==Starch plates==
-
* Pupp (colony1) into the death vector, transformed into TOP10
+
- Use blank plates
-
* Pspac (colony1) into the death vector, transformed into TOP10
+
-
* agrD (colony5) into the death vector, transformed into TOP10
+
-
- Grow this single colony into 10mL of LB (Cm35)
+
- Melt 100mL of Soft Agar, and add slowly 1g of starch
 +
- Mix, boil to sterilize
-
==Transformation of new "biobricks"==
+
- Pour on blank plate
-
- transform into E.coli :
+
- Put 4 or 5 colonies on each plate
-
*agrA
+
 
-
*agrB
+
* IA751 from blank plate (positive control)
-
*agrC
+
* IA771 from blank plate (negative control)
-
*Pxyl
+
* IA751 transformed with ECE112 (Cm5, 29/08)
 +
* IA751 control (Cm5, 29/08)
 +
* IA751 + ECE112 (Cm5, 02/09)
 +
* IA751 + ECE153 (Cm5, 02/09)
 +
* IA751 + ECE153 (Cm5, 29/08)
=September 4th=
=September 4th=
-
==Results of new "biobricks"transformation==
+
==Result of Bacillus transformation (glycerol stock of competent cells)==
-
- Nothing on our plate, except for the control plate.
+
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
! strain !! antibiotic !! added vector !! observation !! conclusion
 +
|-
 +
| IA751 || none || none || confluent colonies || cells alive
 +
|-
 +
| IA751 || Spc50 || none || nothing || no contamination
 +
|-
 +
| IA751 || Spc50 || ECE153 || 15 || ok, efficiency 25 colonies/μg of DNA
 +
|-
 +
| IA771 || none || none || confluent colonies || cells alive
 +
|-
 +
| IA771 || Cm5 || none || nothing || no contamination
 +
|-
 +
| IA771 || Cm5 || ECE166 || 15 || ok, efficiency 5 colonies/μg of DNA
 +
|}
-
==New attempt of new "biobricks" transformation==
+
We have no contamination in our glycerol stocks. However, the efficiency of glycerol stocks seems to be lower than these of fresh competent cells.
-
- transform into E.coli these ligated products:
+
==Result of starch plates==
-
*agrA
+
-
*agrB
+
-
*agrC
+
-
*Pxyl
+
 +
We had 5mL of iodine solution, wait for 1min and observe.
-
=September 7th=
+
Controls (the small zone of clearing are contamination, we have not yet manage to find how to avoid them)
-
==New PCR==
+
Negative control : IA771, no zone of clearing
 +
[[Image:photoesfr.gif|200px |center]]
-
- New stocks of Master Mix
+
Positive control : IA751, big zones of clearing
 +
[[Image:photoesr.gif|200px|center]]
-
- PCR agrA, agrB, Pxyl, Pspac, Ppac and Pupp
+
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
! strain !! vector !! photo !! observation !! conclusion
 +
|-
 +
| IA751 || none || [[Image:photoer.gif|100px|center]] || big zones of clearing for each colony || ok
 +
|-
 +
| IA771 || none || [[Image:photo2.gif|100px|center]] || no zone of clearing for each colony || ok
 +
|-
 +
| IA751 || ECE112/none (29/08) || [[Image:photo3.gif|100px|center]] || clearing zones for control, so no trnasformation, it is maybe E.coli, 1 clearing zone out of 3 colonies, it is certainly contamination, but that means that some colonies are transformed || ok
 +
|-
 +
| IA751 || ECE112 (02/09) || [[Image:photo4.gif|100px|center]] || 1 zone of clearing out of 4 colonies || some colonies are not transformed and have a resistance to Cm5 (contamination?)
 +
|-
 +
| IA751 || ECE153 (28/08) || [[Image:photo5.gif|100px|center]] || no zone of clearing  || 5 transformed colonies
 +
|-
 +
| IA751 || ECE153 (02/09) || [[Image:photo6.gif|100px|center]] || 2 zone of clearing out of 5 colonies || some colonies are not transformed and have a resistance to Spc50 (contamination?)
 +
|}
 +
==Colony PCR from bacillus subtilis colonies==
-
=September 8th=
+
We want to check the size of the insert in the AmyE region(IA751) after transformation with an integration vector (to check if we have a single cross over or not).
-
==Check product of PCR from yesterday==
+
We are going to test our transformation of ECE112 and 153.
-
* Lane3 : Hyperladder I
+
-
* Lane4 : agrA
+
-
* Lane5 : agrB
+
-
* Lane6 : Pxyl
+
-
* Lane7 : Pspac
+
-
* Lane8 : Ppac
+
-
* Lane9 : Pupp
+
-
* Lane10 Hyperladder IV
+
-
[[Image:ge0809pcr.gif|300px|center]]
+
We already tried this protocol, but our PCR did not work, so we tried again, with one sample of chromosomal DNA from bacillus to check if our transformation works.  
-
* Results : everything is ok
+
- add 20μL of 0.05mg/mL lysozyme into a PCR tube
-
==Check transformation from 03/09==
+
- add a few colonies into this tube until you obtain a dense solution. We test :
 +
* IA751
 +
* IA751 + ECE153 (29/08, 02/09, 03/09)
 +
* IA751 + ECE112 (02/09)
-
After several days of incubation, we have a few colonies on our transformed plates (agrA, B and C).
+
- 15min in 37°C, then 15min in 99°C, 1min in 4°C, 1min in 99°C, 1min in 4°C
-
* Single colony PCR
+
- For IA751, IA751 + ECE112, IA751 + ECE153, and  bacillus chromosomal DNA, add 12μL of SDW, 5μL of MM, 1+1μL of primers (amyE primers), 1μL of the solution prepared befor with cells (or DNA)
-
- Add 13μL of cells diluted into SDW, 5μL of MM, 1μL of VF and 1μL of VR
+
- PCR
-
* Gel
+
- Gel
 +
* Lane1 : hyperladderI
 +
* Lane2 : IA751 + ECE153 (29/08)
 +
* Lane3 : IA751 + ECE153 (02/09)
 +
* Lane4 : IA751 + ECE153 (03/09)
 +
* Lane5 : IA751 + ECE112 (02/09)
 +
* Lane6 : IA751
 +
* Lane7 : Chromosomal DNA
 +
[[Image:photoed.gif|300px|center]]
 +
- Result : only chromosomal DNA worked. That means that our primers are ok, but we have to find a protocol for chromosomal DNA miniprep of bacillus to be able to PCR something into the genome of bacillus.
 +
*New attempt without the lyse process (only protocol of single colony PCR, like for E.coli)
 +
- Gel
 +
* Lane1 : hyperladderI
 +
* Lane2 : IA751 + ECE153 (29/08)
 +
* Lane3 : IA751 + ECE153 (02/09)
 +
* Lane4 : IA751 + ECE153 (03/09)
 +
* Lane5 : IA751 + ECE112 (02/09)
 +
* Lane6 : IA751
 +
* Lane7 : Chromosomal DNA
 +
[[Image:photoefr.gif|300px|center]]
 +
- Result : Lane4, we have something but too small.
-
<!-- ## Do not edit below this line unless you know what you are doing. ## -->
+
=September 5th=
 +
==Starch plates==
 +
 
 +
Prepare some starch plates. We want to test our transformation but we can not manage to PCR Bacillus. So we are going to try to extract chromosomal DNA from Bacillus. We make these starch plates to test it with colonies which have positive control with amylase test.
 +
 
 +
- Prepare starch plates (you can find the protocol [[IGEM:Cambridge/2008/Turing_Pattern_Formation/Experiments/Bacillus_subtilis_transfomation#ECE153|here]])
 +
 
 +
- Plate 4 different plates :
 +
 
 +
* 2 colonies of IA75 (positive control) and 3 colonies of IA771 (negative control)
 +
* 5 colonies of IA751 transformed with ECE153 from 29/08
 +
* 3 colonies of IA751 transformed with ECE153 from 02/09 and 3 colonies from 03/09
 +
* 5 colonies of IA751 transformed with ECE112 from 02/09
 +
 
 +
 
 +
=September 6th=
 +
 
 +
==Results of starch plates==
 +
 
 +
We had 5mL of iodine solution, wait for 1min and observe.
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
! strain !! vector !! photo !! observation !! conclusion
 +
|-
 +
| IA751 || none || [[Image:photo06/09controls.gif|100px|center]] || big zones of clearing for IA751 (right), no zone of clearing for IA771 (left) || IA751 : positive control and IA771 : negative control
 +
|-
 +
| IA751 || ECE153 from 02/09 (on left) || [[Image:photo06/091530203.gif|100px|center]] || colony 1 : zone of clearing, no transformation; colony 2 and 3 : no zone of clearing, transformation ok|| 2 transformed colonies out of 3
 +
|-
 +
| IA751 || ECE153 from 03/09 (on right) || [[Image:photo06/091530203.gif|100px|center]] || colony 1,2 and 3 : no zone of clearing, transformation ok|| transformation ok
 +
|-
 +
| IA751 || ECE153 (28/08) || [[Image:photo06/0915328.gif|100px|center]] || no zone of clearing for colonies 1, 2 and 3, impossible to say for colonies 4 and 5 because big contamination (so big zone of clearing) || colony 1,2,3 : ok, no result for the others
 +
|-
 +
| IA751 || ECE112 (02/09) || [[Image:photo06/0911202.gif|100px|center]] || no zones of clearing || transformation ok
 +
|}
 +
 
 +
 
 +
=September 8th=
 +
 
 +
==Prepare chromosomal DNA extraction from Bacillus==
 +
 
 +
- Grow 2 tubes into LB for
 +
* IA751
 +
* IA751 + ECE153 from 03/09 (for tissue kit)
 +
* IA751 + ECE153 from 02/09
 +
* IA751 + ECE112 from 05/09
 +
 
 +
==PCR AmyE from ECE112==
 +
 
 +
- Add 12μL of SDW, 5 μL of MM, 1μL + 1μL of each primers (for AmyE back and AmyE front) and 1μL of ECE112
 +
 
 +
* Lane1 : HyperladderI
 +
* Lane4 : AmyE front
 +
* Lane5 : AmyE front
 +
* Lane6 : AmyE back
 +
* Lane7 : AmyE back
 +
 
 +
[[Image:gelsdgsdg.gif|300px|center]]
 +
 
 +
* Results : good band for AmyE front, very low band for AmyE back
 +
 
 +
 
 +
=September 9th=
 +
 
 +
==Chromosomal DNA extraction==
 +
 
 +
- ZR soil microbe DNA kit for
 +
*IA751
 +
*IA751+ECE112
 +
*IA751+ECE153
 +
 
 +
- Nanodrop
 +
 
 +
{|class="wikitable" style="text-align:center" border="1"
 +
|-
 +
! product (chromosomal DNA) !! concentration (ng/μL)
 +
|-
 +
| IA751 || 75
 +
|-
 +
| IA751 + ECE112|| 48.6
 +
|-
 +
| IA751 +ECE153 || 74.2
 +
|}
 +
 
 +
 
 +
- PCR with AmyE detect primers (12μL of SDW, 5μL of MM, 1+1μL of each primer, 1μL of chromosomal DNA or plasmid)
 +
 
 +
- Gel
 +
 
 +
* Lane1 : HyperladderI
 +
* Lane2 : AmyE Back
 +
* Lane3 : AmyE Front
 +
* Lane4 : Bacillus chromosomal DNA (given by Duncan to check)
 +
* Lane5 : ECE112 (plasmid)
 +
* Lane6 : ECE153 (plasmid)
 +
* Lane7 : chromosomal DNA IA751 + ECE112
 +
* Lane8 : chromosomal DNA IA751 + ECE153
 +
* Lane9 : chromosomal DNA IA751
 +
 
 +
[[Image:photosdfscontrols.gif|300px|center]]
 +
 
 +
* Results
 +
 
 +
- Only something for IA751, and ECE153 (pure plasmid)???
 +
 
 +
- New PCR with 4μL of chromosomal DNA and new PCR program
 +
 
 +
- New gel
 +
 
 +
* Lane1 : IA751 (chromosomal DNA)
 +
* Lane2 : ECE153 (chromosomal DNA)
 +
* Lane3 : ECE112 (chromosomal DNA)
 +
* Lane4 : ECE112 (plasmid)
 +
* Lane5 : ECE153 (plasmid)
 +
* Lane6 : Bacillus chromosomal DNA (given by Duncan)
 +
* Lane7 : HyperladderI
 +
* Lane8 : agrA
 +
* Lane9 : agrB
 +
 
 +
*Result
 +
 
 +
[[Image:photos09092.gif|300px|center]]
 +
 
 +
- No insert in IA751 transformed with ECE153 and ECE112 (same size than control!) and however the amylase test is positive for this colony....
 +
 
 +
==PCR AmyE again==
 +
 
 +
- 24μL of SDW, 10μL of MM, 2μL of each primers, 2μL of ECE112
 +
 
 +
* Results (cf gel, lane 2 and 3)
 +
 
 +
- Very good result for amyE front, nothing for AmyE back
 +
 
 +
=September 10th=
 +
 
 +
==Check PCR for pSBINT1 vector==
 +
 
 +
- Gel
 +
 
 +
*Lane2 : HyperladderI
 +
*Lane3 : rep+bla
 +
*Lane4 : PCR of I714062
 +
*Lane5 : PCD of J04630
 +
 
 +
* Result
 +
 
 +
[[Image:gel11093.gif|200px|center]]
 +
 
 +
- One band for rep, but not very clear, we should try again
 +
 
 +
- for the other parts, we have a good band but the wrong size, these biobricks seem to be bad, we will order them from the MIT
 +
 
 +
=September 11th=
 +
 
 +
==PCR for pBSINT1 biobrick vector==
 +
* PCR rep+bla part from I732007 (pSB1A3 backbone)
 +
 
 +
- Add 12μL of SDW, 5μL of MM, 1μL of rep fwd primer, 1μL of rep rev primer and 1μL of I732007 (program iGEM new)
 +
 
 +
* PCR I714062 and J04630 with VF and VR. We want to extract the cutting sites of the biobrick vector with GFP inside (in the future we would prefer to make our biobrick vector with a RSB for B.S. inside)
 +
- Add 9μL of SDW, 5μL of MM, 1μL of VF, 1μL VR and 4μL of each biobrick (program iGEM new)
 +
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Latest revision as of 03:56, 30 October 2008

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Contents

July 22nd

We have 4 tubes from last year. These strains are frozen in glycerol.

  • PNZ8901 plasmid in E.coli MC1061 Strain
    • Chloroamphenicol resistant
    • "sure" vector
    • does not integrate
  • B. subtilis strain 1A1
    • No resistance
    • Same as strain 168 but tryptophan deficient
  • E.coli strain MC1061, no plasmid
  • PP182 plasmid in E.coli strain DH5alpha
    • Ampicillin resistance
    • integrates


Checking antibiotic resistance

Purpose : Grow each of them on a plate to test their antibiotic resistance

Protocol

  • Warm up frozen tubes
  • Take 15uL of each and put it on plates without antibiotic
  • Incubate at 37°C



July 23rd

  • Take one colony from each of the plates grown from the tubes yesterday and plated them again on LA without antibiotics


1/ Purpose Check antibiotic resistance of our different strains

Strain Plasmid Extracted from Antibiotic use Concentration of antibiotic
DH5alpha PP182 tube Amp 100
MC1061 NONE tube Cm 35
MC1061 PNZ8901 tube Cm 35
DH5alpha PP182 plate Amp 100
MC1061 NONE plate Cm 35
MC1061 PNZ8901 plate Cm 35


2/ Purpose : Find the right concentration of antibiotic so that B. subtilis survive

- Grow 15uL of B. 1A1 (frozen tube) in 5mL of LB

- Incubate at 37°C

3/ Purpose : Grow plasmids in TOP10, transformation

4 plasmids :

  • I746000
  • I746100
  • I746101
  • I746001

1 control : PUC19

- Add 20uL of TOP10 competent cells and 0.5uL of Plasmid in an eppendorf

- 30min on ice

- Heat shock : 60s at 42°C

- 2min on ice

- Add 89.5uL of SOC

- 60min at 37°C

- Put the mix on plate with ampicillin resistance

- Incubate at 37°C


July 24th

  • Test of antibiotic resistances of strains of last year
Strain Plasmid Extracted from Antibiotic use Observations Conclusion
DH5alpha PP182 tube Amp Many colonies Amp Resistance OK
DH5alpha PP182 plate Amp Many colonies Idem
MC1061 NONE tube Cm Nothing As expecting, no Cm Resist.
MC1061 NONE plate Cm Nothing Idem
MC1061 PNZ8901 plate Cm Maybe a few colonies Contamination??
MC1061 PNZ8901 tube Cm No colonies no Cm Resist.


  • Transformation of E.coli with different plasmids from last year
Strain Inserted plasmid Antibiotic Observation Conclusion
E.coli I746000 Amp No colonies Antibiotic resistance unknown, no Amp Resist., Pb : no terminator
E.coli I746100 Amp No colonies Antibiotic resistance unknown, no Amp Resist., Pb : no terminator
E.coli I746001 Amp Many colonies Transformation OK
E.coli I746101 Amp Many colonies Transformation OK

Antibiotic test for Bacillus resistance

We want to find the lowest concentration of antibiotic which kills Bacillus.


Dilution of Amp. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ]

Concentration (μg/mL) 100 75 50 25 10
100 mg/mL Stock 1:1000 3:4000 1:2000 1:4000 1:10000

Dilution of Cm. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ]

Concentration (μg/mL) 35 25 15 10 5
100 mg/mL Stock 1:1000 1:1400 3:7000 1:3500 1:7000
  • Add Disks in antibiotic solution for 5 mins [ Protocol to sterilize tweezers : Wipe with Kimwipes, Ethanol, Flame ]
  • Melt Soft Agar
  • 3mL SA + 10μL Cells 1A1 from LB prepared on 23/7/08
  • Pour SA+Cells over blank hard agar plates
  • put disks with different concentration of Amp/Cm above SA
  • Incubate at 37°C

Results [Plates were prepared before lunch, at 5pm there were visible growth]

-Amp Plates: Huge rings of no growth around 100, 75, 50, 25, and 10.

-Cm PLates: Tiny rings of no growth around 5, 10, 15 and 25. Small ring of no growth around 35 but not well defined. (No clear zones)


Salts for making Bacillus Competent

10x Medium A Base

  • Yeast Extract 10g
  • Casamino Acid 2g
  • add Distilled water to 900mL

-Aliquot into 5 different bottles. [180mL each]

10x Bacillus Salts

  • NH4)2SO4 20g
  • K2PO4 anhydrous 139.66g
  • KH2PO4 60g
  • Tri-Sodium Citrate 10g
  • MgSO4.7H2O 2g
  • Add Distilled water to 1000mL

-Aliguot into 5 different bottles. [200mL each]

Medium B

    • Preparing 50mM CaCl2.2H2O
      • CaCl2.2H2O 1.470g
      • Add Distilled water to 20mL [Final conc. 500mM]
      • Take 10mL of 500mM
      • Add 90mL of distilled water [Final conc. 50mM]
    • Preparing 250nM MgCl2.6H2O
      • MgCl2.6H2O 0.508g
      • Add Distilled water to 10mL [Final conc. 250mM]
      • Take 1mL of 250mM
      • Add 99mL of Distilled water [Final conc. 250μM]
      • Take 1mL of 250μM
      • Add 99mL of Distilled water [Final conc. 250nM]
  • Add 100mL of 50mM CaCl2.2H2O
  • Add 100mL of 250nM MgCl2.6H2O

-Total volume 200mL

NOTE: To complete Medium B, Take 0.2mL of this solution

July 25th

Results from Yesterday

  • Antibiotic test

Results for Amp

Even with a concentration of 10μg/mL, Amp kills Bacillus. Since we want to know which is the lowest concentration of Amp which kills B.S, we are going to test with some lower concentrations.

Results for Cm

Even with a concentration of 35μg/mL, ther is not a clear area around the antibiotic disk. So we have to test some higher concentration of Cm.

Wet Work

  • New antibiotic tests

Dilution of Amp

Concentration (μg/mL) 10 7.5 5 2.5 1
10 μg/mL Stock (from yesterday) 1:1 3:4 1:2 1:4 1:10


Dilution of Cm

Concentration (μg/mL) 35 37.5 40 42.5 45
35 μg/mL Stock (from yesterday) 1:1 5:6 (from 45μg/mL) 1:875 17:18 (from 45μg/mL) 9:7000

- Melt Soft Agar - 3mL SA + 10μL Cells 1A1 from LB prepared on 23/7/08 - Pour SA+Cells over blank hard agar plates - Put 5 blank disks on each agar plate - Add 40μL of antibiotic on each disk - Incubate at 37°C

Results

Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them!

July 28th

  • Results of antibiotic plates from yesterday

For Cm, 35μg/mL should be enough to kill B.S.

For Amp, nothing can be concluded!

Wet Work

  • Check plasmid ppL82

We had 2 samples of ppL82 in DH5α in LB solution (~4mL), one from the frozen glycerol tube and one from a colony picked on a plate. Normally plasmid and cells should not be kept in freezer. So, we want to extract this plasmid, check its size and then keep it in the fridge. We do that for the 2 different samples.


- Plasmid Miniprep (standard protocol)

- Test the concentration of DNA in each tube

  1. ppL82 plate : 89.8ng/mL
  2. ppL82 tube : 71ng/mL
  • Preparation of different stocks of strains and plasmids


PNZ8901

- Plate a single colony (from 23/07/08) on a Cm plate

- Incubate at 37°C


- Grow the entire frozen glycerol tube in 20mL of LB without antibiotic to check if this stock is still good (we will check the plasmid on a gel tomorrow)

- Incubate at 37°C


MC1061

-Pick e single colony of MC1061 (from 22/07/08) and put it in 10mL of LB (to make glycerol stocks)

-Incubate at 37°C


1A1

- Add 100μL of LB+1A (mix of friday) and 10mL of LB

- Incubate at 37°C


  • Make B.S. 1A1 competent and transform them

- Prepare Medium A

Add 81mL of SDW, 10mL of 10X Medium A base and 9mL of 10X Bacillus salts


- Make B.S. 1A1 competent

  1. In 10mL of medium A and add about 10 colonies of B.S.
  2. Check the OD650, you should have an OD between 0.1 and 0.2. t0 for OD = 0.1876
  3. Check the OD650 every 20min and plot OD against time on semi-log paper. When the point at the culture leaves log growth, it is ok
Time (min) 0 20 40 60 70 80
OD650 0.1876 0.2074 0.3282 0.4545 0.4895 0.5040
Log of OD650 versus time for Competent Bacillus Cell Culture

At, t0 = 70min, log growth seems to stop.

  1. Incubate at 37°C 90min after t0
  2. Warn 10 Ependorf tubes with 0.45mL of Medium B
  3. Add 50μL of culture in each tube of Medium B at t90
  4. Incubate the diluted culture at 37°C with vigorous aeration for 90min

- Storage : we want to store 7 tubes

6 tubes in the freezer (with 60μL of glycerol)

1 tube on the bench

- Transformation

  1. We make ppL82 from plate, ppL82 from tube, and a control
  2. Add 0.5μg of DNA (15μL)
  3. Incubate at 37°C for 30min
  4. Plate on Cm plates


July 29th

  • Result from the gel (29/07/2008)
  • Lane8 : ppL82 (plate)
  • Lane9 : ppL82 (tube)
  • Lane 10 : I746001
  • Lane 11 : I746101
  • Lane 12 : hyperladderI
Photoa.gif

The ladder seems to be wrong. So it was really difficult to ckeck the size of our plasmid ppL82. To make, that, we assume that the size of our biobricks was ok, and we estimate the size of our plasmid. The plasmid ppL82 seems to have the right size, but we will have to check again to be able to use a ladder.

  • Competence of B.S. kept on the bench

We observed B.S. with a microscope. Problem, all B.S. seems to be dead!

Possible reasons of this problem :

- Problem with the vector (it could be a wrong vector, we are not really sure)

- Quantity of DNA, we added 0.5μg. The protocol was 1μg, but normally it should be ok.

- B.S. may not be competent (we have to test motility with microscope)

- Cells could be dead : replate them

- Try to add tryptophan in the medium


Probable reason : we forgot to dilute the medium B, that's why it did not work


  • Results from transformation plates (B.S.)


There are 2 colonies on a plate, but it does not look like Bacillus. Moreover, there is also a colony on our negative control plate. SO it must be contamination (resistant to CM!). We will do the transformation again.


Wet Work

  • Prepare medium for transformation of B.S.


- Preparation of medium B: 10mL of medium A and 0.2mL of 50mMCaCl22(H2O) + 250mMgCl26(H2O)

- Preparation of medium A with tryptophan : 81mL of SDW, 9mL of 10X Bacillus salts, 10mL of 10X Medium A base and 0.1mL of Tryptophan (11mg/mL)


  • Check plasmid PNZ8901


- Plasmid miniprep (same protocol with 60μL of elution buffer)

- Digest : with PstI and SalI from Biolabs, and Buffer 3 (add 15μL of DNA)

- Gel (17μL of sample and 3μL of dye)


Results from the gel

  • Lane9 : PNZ8901
  • Lane10 : HyperladderI


Photoh.gif

The sizes we expected were about 1100kb and 2100kb. The sizes should be ok!


July 30th

  • Transforming Bacillus Subtilis with medium A and medium A + tryptophan

Medium A

Time (min) 0 20 40 60 73 85 95 105
OD650 0.1394 0.1370 0.1660 0.2304 0.2778 0.3138 0.3373 0.3583

For this medium, the curb stop to increase logarithmically at 100min.


Medium A + tryptophan

Time (min) 0 20 40 60 73 85 95
OD650 0.1493 0.1636 0.2080 0.2909 0.3512 0.4002 0.4165

For this medium, the curb stop to increase logarithmically at 90min.


- Incubate 90min at 37°C by shaking

- Prepare 7 tubes for medium A and 7 tubes for medium A + tryptophan : add 0.45mL of prewarmed medium B and 0.05mL of culture and incubate 90min at 37°C by shaking.

- Transform B.S. by adding DNA and incubate 30min at 37°C

We have one transformation with ppL82 (add 7.5μL of DNA), one with PNZ8901 (add 10μL of DNA) and one negative control without DNA.

- Plate 500μL on Cm plate (35μg/mL)


- For the remaining tubes, in one, add 60μL of glycerol and keep it in the freezer and keep the other one on the bench (for each medium)

  • Checking our big stock of biobricks and PNZ plasmid


We did big culture of biobricks and PNZ8901 plasmid to be able to make stocks. We want to check them before making stocks.

- Plamsid miniprep (for I746001, I746101 and PNZ8901 from big flasks

- Measure DNA concentration in our samples to decide the volume of DNA we have to add in our preparation

260/280 DNA concentration (ng/nl)
I746001 1.79 50.5
I746101 1.87 54.3
PNZ8901 1.84 87


- Digest

For I746001 and I746101 For PNZ8901 plasmid
14μL of SDW 14μL of SDW
2μL of 10X Fast Digest Buffer 2μL of Buffer 3 (Biolabs)
2μL of DNA of biobricks 2μL of DNA of PNZ8901
1μL of EcoRI 1μL of PstI
1μL of SpeI 1μL of SalI


- Gel

  • Results from this gel
  • Lane 9 : I746001
  • Lane 10 : I746101
  • Lane 11 : PNZ8901
  • Lane 12 : HyperladderI
Photob.gif

Everthing is really too big! There is a problem, either dimerization, either contamination, either a problem in our work. So we are going to run a new gel.

  • New gel to check

- Miniprep plasmid from growth bottles (I746001, I746101 and PNZ8901); from plates (I746001, I746101 and PNZ8901).

- Do single digest for PNZ8901 (one with PstI, one with SalI)

- Run a gel with : PNZ8901 from friday, PNZ8901 digest with PstI, PNZ8901 digest with SalI, 3 samples from growth bottles, 3 samples from plates (to check the size of the uncut vectors)

  • Result from this second gel
Photoc.gif

The ladders are really bad! But the size of our biobricks and plasmid are too big! So we can not trust these big cultures. They is a problem. With tests from the first week, we know that biobricks are right, so we are going to grow new cultures from these first cultures, to make stocks.

Concerning the vectors, they are from last year, so we are not sure of what they are. Since we ordered new well defined vectors (we should receive them on friday), we will use them in the next steps to be sure of our work.


August 1st

  • Grow received vectors
Vectors AmpR (μg/μL) CmR (μg/μL) KanR (μg/μL)
ECE112 100 0 0
ECE147 50 0 0
ECE149 50 0 0
ECE150 50 0 0
ECE151 50 0 0
ECE153 100 0 0
ECE162 100 0 0
ECE165 100 10 0
ECE166 100 10 0
ECE171 50 0 10
ECE172 100 10 0
ECE176 100 5 0

- Preparation of Agar plates

We have bottles of 200mL.

Type Amp (μL) Cm (μL) Kan (μL)
Amp100 200 0 0
Amp50 100 0 0
Amp100 + Cm10 200 57.1 0
Amp100 + Cm5 200 28.6 0
Amp50 + Kan10 50 0 80


- Preparation of LB tubes

We prepare 10mL of LB in which we add a single colony.


Type Amp (μL) Cm (μL) Kan (μL)
Amp100 10 0 0
Amp50 5 0 0
Amp100 + Cm10 10 2.9 0
Amp100 + Cm5 10 1.4 0
Amp50 + Kan10 5 0 4


August 2nd

Yesterday's Results

  • All Plates grew!
    • ECE 172 seemed to have trouble growing on the plate (no single colonies, just one big lump at the start of the streak)
    • ECE 150 grew too well! (possibly hard to pick out single colony)
  • Tubes:
    • ECE 176, 172, 153 seem to be quite clear!!
    • ECE 150, 151 had strange floating 'colonies' in the tube
    • All other tubes were quite cloudy

Lab Work

Fridged all plates except ECE 172 ==> Benched.

All tubes palleted and placed in freezer although ECE 176, 172, 153 doesn't seem to have any pallets

All red plates does not seem to have any extra growth despite the air conditioning had been turned off when I entered the lab this afternoon.


August 4th

Wet Work

  • Check vectors

- for each vector, plasmid miniprep from frozen pellets (step 7 only once, with 60μL of Elution Buffer for ECE112, ECE147, ECE149, ECE 150 and only 30μL for the others)

- Single digest (15μL of SDW, 2μL of Buffer, 2μL of DNA, 1μL of enzyme, then incubate 10min at 37°C and 30 min for the one using HindIII with EcoRI buffer)

- Double digest (mix 7.5μL of each single digest, and then incubate 10min (or 30min for EcoRI + HindIII and EcoRI buffer) at 37°C, then heat shock 5min at 80°C

Vectors Enzyme 1 Enzyme 2 Buffer Entire size Size of part1 Size of part2
ECE112 XbaI EcoRI Fast Digest 10156 6900 3295
ECE147 EcoRI HindIII EcoRI 5482 4913 600
ECE149 SpeI PstI Fast Digest 6192 4919 1300
ECE150 SpeI HimdIII Buffer 2 6166 5300 778
ECE151 SpeI EcoRI Fast Digest 6166 4825 1300
ECE153 SalI Buffer 3
ECE162 SalI Buffer 3 7600 6000 1600
ECE165 EcoRI HindIII EcoRI 5952 5200 793
ECE166 EcoRI HindIII EcoRI 7262 5100 2103
ECE171 EcoRI PstI Fast Digest 5444 3600 1826
ECE172 HindIII Buffer 2 6462 3900 2540
ECE176 EcoRI XbaI Fast Digest 866 4250 5128
  • Results
  • Lane1 : HyperladderI
  • Lane2 : ECE112
  • Lane3 : ECE147
  • Lane4 : ECE149
  • Lane5 : ECE150
  • Lane6 : ECE151
  • Lane7 : ECE162
  • Lane8 : ECE165
  • Lane9 : ECE166
  • Lane10 : ECE171
  • Lane11 : ECE172
  • Lane12 : HyperladderI


Photoi.gif

- ECE112, ECE165, ECE166, ECE171 : right bands, ok

- ECE172 : no band, not enough DNA

- ECE147, ECE150, ECE162 : only one band, maybe not enough DNA

- ECE149 : wrong size!

- ECE151 : not sure, check again


  • Plates

- Streak new plates with different strains of Bacillus : 1A1, IA751 and IA771


August 5th

Checking vectors

- Plasmid miniprep for ECE166 (for stock), ECE172 and ECE153

- Digest of ECE153, ECE172 and ECE162 (with plasmid miniprep from yesterday)

- Run on a gel single digest for ECE147 (from yesterday with more DNA), ECE149 (from yesterday with more DNA), ECE150 (from yesterday with more DNA) and ECE 153, ECE162, ECE172

  • Results
  • Lane3 : HyperladderI
  • Lane4 : ECE147
  • Lane5 : ECE149
  • Lane6 : ECE150
  • Lane7 : ECE151
  • Lane8 : ECE1162
  • Lane9 : ECE172
  • Lane10 : hyperladderI


Photoj.gif

Very low bands : not enough DNA!!!

Transformation of Bacillus

  • Result of Nanodrop
Vector 260/280 ng/μL
ECE112 1.75 64.6
ECE166 172 138.6

- Prepare medium A with tryptophan, and medium B

- add 5mL of medium A in 3 different tubes, inoculate each tube with colonies (1A1, IA751 ans IA771)

- Check OD every 20min

- Incubate 90min

- Add 0.45mL of medium B and 0.05mL of culture in Ependorf tubes

- Incubate 90min

- Transform : add 1μg of DNA (some with ECE112, some with ECE166) (so you need to nanodrop samples before!)

- Incubate 30min

- Pipette 200μL of solution, spread it on aech plate, wait 10min, and do it again

- Incubate 24hours

- A few glycerol tubes to stock cells : add 2/7 glycerol to cell tubes


- Transformation from glycerol stock from 30/07/2008

- Spin glycerol stocks, pipette out glycerol

- Add 0.5mL of medium B, incubate for 1 hour

- Add 10μL of ECE112 (640ng)

- Incubate 2hours

- Plate 200μL, and 10min later, still 200μL


New stocks

- Do glycerol stock of I746001 and I746101 (no sterile glycerol)

- Put IA751, IA771 in 10mL LB

- Put ECE 176 in 10mL LB + antibiotic

- Reinoculate the tube of LB from yesterday with ECE166 plate

- ECE176 replated onto Amp100 + Cm5


August 6th

Result from transformation

PLATES HAD BEEN BINNED!! OOPS

Check Vectors

- Plasmid miniprep for ECE176 (from 05/08 LB stock and from 04/08 LB stock with a second inoculation)

We want to check uncut plasmid, single and double digest for ECE147, ECE149, ECE150, ECE153, ECE162, ECE172, ECE176. In order to have enough DNA (to see the bands), we will add 1μg of DNA to make single digest, and wr will incubate during 1h (instead of only 10min).

  • Nanodrop
Vetor 260/280 μg/mL
ECE147 1.67 50.8
ECE149 1.66 51.9
ECE150 1.78 107.0
ECE153 1.68 31.2
ECE162 1.56 33.1
ECE172 (04/08) 1.64 22.8
ECE172 (05/08) 1.89 213.1
ECE176 (04/08) 1.89 111.1
ECE176 (05/08) 1.83 96.2

We made singe digest for the samples from 05/08 only.

- Double digest (1 hour of incubation in water bath at 37°C)

- Gel

Gel1

  • Lane1 : HyperladderI
  • Lane2 : ECE147 with EcoRI
  • Lane3 : ECE147 with HindIII
  • Lane4 : ECE149 with SpeI
  • Lane5 : ECE149 with PstI
  • Lane6 : ECE150 with SpeI
  • Lane7 : ECE150 with HindIII
  • Lane8 : ECE153 with SalI
  • Lane9 : ECE162 with SalI
  • Lane10 : ECE172 with HindIII
  • Lane11 : ECE176 with EcoRI
  • Lane12 : HyperladderI
Photok.gif

Gel2

  • Lane1 : HyperladderI
  • Lane2 : ECE176 with XbaI
  • Lane3 : ECE176 double digest
  • Lane4 : ECE150 double digest
  • Lane5 : ECE149 double digest
  • Lane6 : ECE147 double digest
  • Lane7 : HyperladderI
  • Lane8 : ECE149 uncut
  • Lane9 : ECE153 uncut
  • Lane10 : ECE172 uncut
  • Lane11 : supercoiled ladder
Photol.gif
  • Results

Our supercoiled ladder was very bad, so it was impossible to conclude for uncut vectors.

- ECE147, ECE 150, ECE153, ECE 176 single digest : ok

- ECE149 : problem, 2 cutting sites for PstI (only one in the sequence)

- ECE162 : only one band whereas SalI should have 2 cutting sites

- ECE172 : wrong sizes!!!

- Double digest for ECE176 : ok!

- Double digest for ECE 150, 149 and 147 : only one band!!!

There may be a problem when we do the double digest by adding two single digest. We will try again with a direct double digest.

August 7th

Transformation of B.S. IA771

New transformation with the same protocol than 2 days ago. We will try to add less liquid on plates (to avoid growing colonies in liquid)

Tube Plate 1 Plate 2 Plate 3 Plate 4
ECE166 Cm5 + 200μL of cells Cm5 + 100μL of cells Cm5 + 50μL of cells Cm10 + 100μL of cells
ECE153 Spc50 + 200μL of cells Spc50 + 100μL of cells Spc50 + 50μL of cells
ECE166 Cm5 + 100μL of cells Cm10 + 100μL of cells
ECE153 Spc50 + 100μL of cells
no DNA Cm5 + 100μL of cells Cm10 + 100μL of cells Spc50 + 100μL of cells
ECE166 (glycerol stock) Cm5 + 100μL of cells Cm10 + 100μL of cells

Check Vectors

We want to make a final check for ECE147, 149, 150, 153 and 162.

- Double digest + gel

  • Results
  • Lane3 : HyperladderI
  • Lane4 : ECE147
  • Lane5 : ECE149
  • Lane6 : ECE150
  • Lane7 : ECE153
  • Lane8 : ECE162
  • Lane9 : HyperladderI
Photom.gif

- ECE 147, ECE150, ECE153 :ok!

- ECE 149 : 3 bands! not the right vector

- ECE162 : ony one band! It has been uncut... maybe the good vector (according to precedent gel), but not sure

Test ECE112 transformation

- for the three different strain (1A1, IA771, IA751), make 12 spots

- take one colony with a loop, and put it on a Cm5 plate and on a Cm5 + Spc100 plate (do that for the 12 spots)

- incubate

New stock

- Put ECE171 in 10mL of LB

- Incubate

August 8th

Result from yesterday transformation

Vector Antibiotic Quantity of cells added number of colonies
ECE166 Cm5 200μL 0 : problem!!!
ECE166 Cm5 100μL 2
ECE166 Cm5 50μL 1
ECE166 Cm10 100μL 0 : no resistance to Cm10!
ECE166 (spin) Cm5 100μL a lot, confluent
ECE166 (spin) Cm10 100μL 0
ECE166 (glycerol + spin) Cm5 100μL about 150, very small
ECE166 (spin) Cm10 100μL 0
ECE153 Spc50 200μL 21 + a lot of confluent colonies
ECE153 Spc50 100μL 7 + confluent (a few)
ECE153 Spc50 50μL 4
ECE153 (spin) Spc50 100μL 25 + about 200 (small and almost confluent)
No DNA Cm5 100μL 0
No DNA Cm10 100μL 0
No DNA Spc50 100μL about 12, maybe more : problem!!!!!

Result for test of ECE112 transformation

On Cm5 plates, we have some colonies for each strains, on Cm5 + Spc 100 plates, no colonies on each plates. This result seems good, however, we forgot to make a control. Since, we had some colonies on control plates, it is possible that our bacillus are resistant to Cm5, even if they are not transformed. We addded some control colonies.

Control with erythromycin

- Prepare erythromycin, stock 5mg/mL : 0.05g in 10mL of SDW

- Prepare plates : add 20μL of Ery (5mg/mL) into 200mL of agar (final concentration : 0.5μg/mL)

- Do several spots on each plate

Plate Colonies added from
1 ECE166 + 100μL of cells (2 different plates)
2 ECE153 + 100μL of cells (2 different plates)
3 ECE166 (from glycerol stock)
4 Control (from a plate of IA771 without antibiotic)
5 IA771 + ECE112 (05/08)
6 IA771 + ECE166 (05/08)

Amylase production screening

  • Prepare agar plate with starch

- Starch : 1g of starch in 10mL of SDW

- Add 2mL of Starch solution into 200mL of agar, mix

-Inoculate on different plates : 1A1 +ECE112 (plate from 05/08), IA751 + ECE112 (plate from 05/08) and IA771 + ECE153

New stocks

- Take 1mL from LB stock oh ECE171 (from 07/08) and put it into 99mL of LB

August 11th

Erytromycin Experiment

- New preparation of Ery : 0.05g into 10mL of ETHANOL

- We plated again our plates (same than 08/08/08)

Amylase screening experiment

- New method : add 2g of starch powder into 200mL of Agar, shake, pipette to plate (and avoid bubbles)

- Same plates than 08/08/08

Test our stock of ECE171

-Plasmid miniprep 9from the samples of 10mL and the one of 100mL)

-Nanodrop both DNA

Vetor 260/280 μg/mL
ECE171 (10mL) 1.73 136.3
ECE171(100mL) 1.82 99.5

- Double digest (EcoRI and PstI) with 10μL of DNA and 1h of incubation

- Samples have been frozen, they should be run onto a gel

Control of Spc resistance of Bacillus

- Spc50 plate with transformed IA771 (ECE153, which is Spc resistant) and with IA771 9which should not be Spc resistant)

August 12th

Results from yesterday

  • Ery plates

- IA751 (control) : most of colonies did not grow, which is good, but 2 or 3 grew...

- IA7771 + ECE166 : all colonies grew : ok 9non integration vector)

- IA771 + ECE153 : growth of all colonies... problem!!! if IA771 is transformed with this integration vector, it should lose its EryR

- IA771 + ECE112 : growth of all colonies... problem!!! if IA771 is transformed with this integration vector, it should lose its EryR


  • Spc resistance

- IA771 : no growth on Spc50 plate, so no natural resistance

- transformed cells grew

  • Amylase plates

- Add iodine solution for 1min

- Problem : no aparent blue, no diffusion of iodine into LA agar + a lot of contamination

New plates and LB stocks

- In order to try to make plasmid miniprep at the end of the day, grow IA771 transformed with ECE166 in 10mL LB + Cm5

7hours is not enough to reach exponential phase!!! We will try again tomorrow by incubating longer

- Grow IA771 and IA751 ( first plate from sterile disks of strain) imto 10mL of LB

- Grow ECE166, 171, 153 in E.coli with approriate antibiotics 9to transform tomorrow)

- Grow IA771 transformed with ECE153 into LB+Spc50 (to check fluorescence with xylose induction)


New test for amylase

We tried 2 different methods.

- Dilute 1g of starch into 100mL of agar and try to dilute it and boil it to sterilize. The problem is that it is very difficult to dilute...

- The second method seem to be better. We diluted 1g of starch into 100mL of Soft Agar (it dilutes very well). Then plate blank agar plate (LA) and then add a thin layer of SA ith 1% of starch. Poke the plates.

- We plated IAI+ECE112, IA751+ECE112 and also IA751 and 1A1 for control.

August 13th

Results for Starch plates

- Add 5mL of iodine

- 1A1 or IA751 : big zones of clearance

- IA751 + ECE112 : no zones of clearance (photos), just small white points on colonies : the gene AmyE seems to be knocked out, transformation ok!

- 1A1 + ECE112 : problem, soft agar melted... impossible to observe!


Transformation of IA751

- We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171

- Spectrophotometer : blank made with medium A

Time (min) 0 20 40 60 80 100 120
OD650 0.1487 0.1541 0.1642 0.1980 0.2470 0.3205 0.3643

- t0 = 120min

- Follow the protocol of transformation

- Plasmid miniprep of ECE153, 166, 171 (to make the transformation)

- Nanodrop ( to add 0.5μg of DNA for transformation)

Vetor 260/280 μg/mL quantity of DNA to add for transformation (μL)
ECE153 1.5 18.4 27.2
ECE166 1.83 39.5 12.7
ECE171 1.83 115.8 4.4

- Plate (with appropriate antibiotics)

  • ECE166 : Cm5
  • ECE153 : Spc50
  • ECE171 : Kan5 (to prepare agar plate, 40μL of Kan 25mg/mL into 200mL of agar)

Check our stock of ECE171

- We made big stocks of ECE171, before keeping it, we wanted to check it, so we run double digest on a gel!!

- Results : problem!!

- 2 possible causes : Double digest was made " days before and kept in the freezer (possible degradation) + too much DNA on the gel

- New double digest (from pellets in the freezer) + new gel

- Results : ok!

Glycerol stocks

- for IA751 and IA771, add 100μL of culture and 500μL of glycerol (60%)

Xylose experiment

- To test the transformation of ECE153, we want to induce the promoter Pxyl, and we will have green fluorescence

- Add 1mL of culture IA751 + ECE153 (from yesterday), 8mL of LB and 45μL of Spc50, 1mL of xylose (1g into 10mL)

Plasmid miniprep for B.S.

- We first tried to use the same protocol than for E.coli (Zyppy kit) for ECE166 transformed in IA751

- Nanodrop to see the result

Vetor 260/280 μg/mL
ECE166, colony 1 1.69 11.2
1.41 14.5
ECE166, colony 2 1.34 7.0

- We have very low concentration of DNA, we will digest that plasmid tomorrow morning to see if it is ECE166, and if it does work, we will try to modify our protocole tomorrow

Transformation of ECE188, 189, 190 in E.coli

  • We received this vector in DNA stocks, so we have to transform them to test them 9because we do not have enough DNA)
  • Use TOP10 competent cells
  • Pellet, add 100μL of CaCl2 solution, for each transformation, use 50μL of cells
  • Add 2μL of DNA, and 1μL of PUC19 for control
  • 30min on ice
  • 2min at 42°C, 2min on ice
  • 2h at 37°C
  • Plate on Amp100 plates

In Preparation of Beta-galactosidase Assay (Promoter Assay PA)

Biobrick Extraction:

E0040

  • GFP (Amp resistance)
  • Extracted from 2007 Plate 1 Well 5H

I13522

  • E.coli constitutive promoter with RBS and GFP (Amp resistance)
  • Extracted from 2007 Plate 3 Well 13C

B0034

  • E. coli RBS (Amp resistance)
  • Extracted from 2008 Plate 1000 Well 2E

R0040

  • E. coli promoter (Amp resistance)
  • Extracted from 2008 Plate 1000 Well 4C

Transformation of competent TOP10 with the 4 biobricks above along with pUC19 control

  • 20μL TOP10 used for each transformation with 2μL DNA
  • Followed standard protocol
  • Plated out neat and 1/10 on Amp100 plates after 2 hours of incubation, then plates are kept at 37°C overnight

August 14th

Results of transformation

Vector Antibiotic number of colonies Transformation efficiency
ECE166 Cm5 31 62
Control Cm5 0
ECE171 Kan5 5 + a lot of small colonies  ?
Control Kan5 1 + 5 very small
ECE153 Spc50 about 25 (on a side) 50
Control Spc50 6 + 2 very small

- Problem with our control, maybe we should check the resistance of competent cells (before transformation)

BioBrick extraction for testing promoters and RBS in B.subtillis

The primers for inducible B.subtillis promoters have been ordered. Meanwhile, we would like to be able to compare the RBS and promoter strengths in E.coli and B.subtillis, using GFP fluorescence to quantify gene expression.

We attempted to isolate 4 BioBricks: I13522 (GFP under constitutive promoter), E0040 (GFP only), R0040 (a promoter), & B0034 (an E.coli RBS). So far, only the former two, extracted from 2007 wells, grew after transformation. Single-colony PCR was used to test the transformants.

Expected VF2-VR fragment sizes:

I13522 - ~2.4 kb E0040 - 958 bp R0040 - 292 bp B0034 - 250 bp

Transformation of vectors 188, 189, 190

- Transformation of yesterday did not work! there was nothing on plates, even on the control plate

- Try again, same protocol, 2μL of DNA, control : PUC9

- Plate on Amp100 for control and Amp100 + Cm5 for vectors

Double digest of ECE166 (extracted from transformed Bacillus yesterday)

- Transformation from 07/08

- 16μL of DNA, 2μL of Buffer EcoRI, 1μL of EcoRI (Biolab) and 1μL of HindIII

- Incubator at 37°C for 35min, heat shock at 80°C for 5min

- Run on a gel with 4μL of DNA

  • Result : no bands! not enough DNA!

- Run on a new gel with 16μL of DNA

  • Result : " bands, one of about 2000b, and one of about 5000b> The big one seem to be too small...

We are going to check this transformation with fluorescence too!

Erythromycin plate

- Erythromycin plate for the transformation 13/08/08

- IA771 (control, they should survive)

- IA771 + ECE166 (non integration vector, so colonies should survive)

- IA771 + ECE171 (integration vector but in another locus, should survive)

- IA771 + 153 9integration vector, they should die if they are transformed)

New stocks

- LB stocks with antibiotic of ECE151, 153, 166

- LB stock of ECE153 with Spc50 (one from colonies from LB stock from 11/08 and one from 13/08 transformation plates)

Check fluorescence with microscope

  • ECE166 from the plate from 13/08

We diluted one colony from this plate into SDW and observed with microscope.

Result : Bacillus is fluorescent! There are some bacteria which are brighter than others, but it is because it is not an integration vector. This transformation worked!!!

  • ECE153

We added xylose, but we did not incubate our sample after that, so it did not work> We will try again tomorrow with a period of incubation>

We will have to

Plates from yesterday for Biobrick Extraction (PA)

  • Growth observed on Amp100 plates for I13522 and E0040 only but not for R0040, B0034 and pUC9 control
  • Re-plated overnight culture neat on Amp 75 plates and incubate at 37°C overnight

Single Colony PCR of I13522 and E0040

  • 5 colonies picked from each neat agar plate onto Amp75 plates
  • PCR following standard protocols
  • Run PCR product on 1.2% agarose gel at 70V which is later soaked in EtBr

First Row:

  • Lane 2 - Hyperladder IV
  • Lane 3-7 - I13522 Colonies 1-5 PCR
  • Lane 9-13 - E0040 Colonies 1-5 PCR

Second Row:

  • Lane 2 - Hyperladder IV
  • Lane 3-6, 9 - R0010 Colonies 1-5 PCR

Result:

  • Expected size of band: I13522 - 2375bp; E0040 - 958bp; R0010 - 292bp
  • Picked colonies 4 and 5 for E0040 into 10ml LB with Amp100 and incubate overnight at 37°C as the band corresponds to about 958bp
  • Band of R0010 colonies 3 and 5 correspond to 292bp and will incubate in LB tomorrow when colonies have grown on agar plates

August 15th

Transformation ECE188, 189, 190 into E.coli

- Results : only our control plate grew!!

- Maybe we did not add enough DNA, or there is another problem. We will e mail the guy from Bacillus center before trying again, because we don't have enough DNA.

Plasmid miniprep ECE 151 (x2), ECE 153, ECE 166

  • Nanodrop results :
260/280 ng/μL
ECE 151 (3) 1.79 86.8
ECE 151 1.88 106.3
ECE 153 1.80 32.2
ECE 166 1.87 36.2


Xylose Induction with ECE 153

- 4 Tubes

- No fluorescence! We have to think about the transformation of integration vectors into Bacillus.


Glycerol Stock Transformation

- Spin down cells (competent) from 5/8 and 13/8 stocks

- Remove liquid, resuspend in Medium B

- Incubate for 60 mins at 37°C

- Add ECE 166 into tubes

- Incubate for 30 mins at 37°C

- Plate out on CM 5 plates

- 4 plates

  • 771 (5/8) + ECE 166
  • 771 (5/8) Control
  • 771 (13/8) + ECE 166
  • 771 (13/8) Control


Transforming ECE 188, 189, 190 (3rd try)

- 2 tubes of chemically competent top 10

- Spin down, remove liquid

- Resuspend cells in 100μL of CaCl2 50mM

- 4 tubes with 50μL of cells

  • ECE 190 (all)
  • ECE 189 (all)
  • ECE 188 (all)
  • PUC 9 (5μL) [ Control ]

- Ice for 30 mins

- 42°C for 2 mins

- Ice for 2 mins

- Incubate at 37°C for 2 hours

- Plate out on Amp 100 (Neat)

Biobrick E0040 and I13522 (PA)

  • PCR E0040 colonies 4 and 5 after miniprep of plasmid to ensure plasmid identity again
  • Protocol:
    • SDW 7.5μL
    • MasterMix 10μL
    • VR and VF2 1x2μL
    • DNA sample 0.5μL
  • Run on 1.2% SyBr E-gel
  • Lane 2 - 100bp ladder
  • Lane 3 - E0040 colony 4
  • Lane 4 - E0040 colony 5
  • Result: Bands at around 1000bp on both lanes 3 and 4 with a brighter band for lane 3. Correct band as E0040 VF-VR is 958bp
  • Minprep of plasmid E0040 from colony 4 is kept
  • Transform chemically compotent TOP10 with I13522 biobrick extract again
  • Transformation following standard protocols
  • TOP10 with I13522 plated neat and 1/10 on Amp. 100 plates and incubate at 37°C overnight
  • Will pick single colonies tomorrow for PCR analysis and into LB with Amp100

August 16th

Result from the days before

  • For Ery plates

- Everything grew, we will try again to see if there is a problem with the antibiotic (and we will do a negative control with the strain IA751)

  • Transformation of E.coli with ECE188, 189, 190

- We have a result from our 3 transformation in E.coli. To transform E.coli with these vectors we have to wait for 2 days before seeing cells!! We will check the vectors next week.

  • Transformation of B.S. IA771 (glycerol stocks)

- Nothing! Problem with the storage of competent IA771???

August 18th

Test Ery efficiency

Since all Ery plates grew, we want to check the antibiotic stock.

- New Ery plate of IA751 (they should die)

- New LB stock (Ery5) with IA751

Single colony plates

- New plates (Cm5 + Amp100) with single colony of ECE188 (from 14/08), ECE189 (from 13/08) and ECE190 (from 14/08)

- Grow the same single colony into LB with antibiotic

Transformation of glycerol stocks of B.S.

The result from the last transformation of glycerol stocks was negative> We tried again with two different protocols.

  • First protocol

- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B

- Incubate about 1h15

- Add 0.6μg of ECE166 (because we managed to transform BS with this vector)

- Incubate 1h30

- Plate on Cm5 plates (DNA less control and transformed cells)

  • Second protocol

- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B

- Add 0.6μg of ECE166 (because we managed to transform BS with this vector)

- Incubate 30min

- Plate on Cm5 plates (transformed cells)


- Plate cells after 10min of incubation (without DNA) on blank plates (to see if they are alive)

PA with I13522, E0040 and B0034 Preparation

  • R0040 TOP10 miniprep from LB culture done by Kevin
  • 5 single colonies of I13522 picked onto Amp100 plate and LB with Amp100 done on Sunday
  • Miniprep of the 5 colonies with sinigle colony PCR
  • PCR followed standard protocol and product is run on 0.8% EtBr E-gel
  • iGEM34 porgramme is used for PCR

Gel:

  • 4μL dye with 20μL PCR product
  • 5μL Hyperladder I with 15μL SDW into lane 2
  • Lanes 3-7: I13522 Colonies 1-5
  • Lane 8: R0040
  • Lane 9: Hyperladder I

Result:

  • Size of I13522 is not right as the bands are all around 4500bp but it should be 2375bp
  • R0040 is of the correct size
  • Transformed chemically compotent TOP10 with B0034 biobrick extract and pUC9 control
  • Plate on Amp100 plates after standard transformation

August 19th

Results from the transformation with glycerol stocks

We used two different glycerol stocks, one from 07/08 and one from 13/08. We also had 2 different protocols (cf 18/08 page).

We tested our stocks on blank plates, cells are alive, the problem is to know if they are still competent or not.

  • Protocol 1 (with incubation time before adding DNA)

- Stock from 07/08 : nothing on the control and nothing on the transformation plate.

- Stock from 13/08 : nothing on the control and nothing on the transformation plate.

  • Protocol 2 (without incubation time before adding DNA)

- Stock from 07/08 : nothing on the control and nothing on the transformation plate.

- Stock from 13/08 : nothing on the control, and 1 colony on the transformed plate. We checked the fluorescence of bacteria into this colony (vector ECE166 with promoter and GFP). We have fluorescence, so our transformation is ok.

The result of this confirm that our stock of competent cells in glycerol is still competnet since we managed to transform one colony. The problem is certainly not the storage, but our protocol when we use frozen competent cells> We need to improve the efficiency of this protocol.

Test our Erythromycin stock

- We put some colonies on a Ery 0.5 plate. They grow, but in a very small amount, so our antibiotic seems to be fine.

- Cells in LB with antibiotic : no growth!

So our antibiotic is ok, either we did not transform our cells and that is why everything grew on Ery plates, either there is a problem in our protocol for Ery test.

Plasmid miniprep of ECE153, ECE166 and ECE171

- Plasmid miniprep LB stocks from yesterday

- Nanodrop

260/280 ng/μL
ECE 153 1.63 32.6
ECE 166 (1) 1.73 56.2
ECE 166 (2) 1.67 57.7
ECE 171 1.81 185.5

Transformation of IA771

We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171.

Spectrophotometer : blank made with medium A

Time (min) 0 20 40 80
OD650 0.1512 0.1498 0.1476 0.1535

There was a problem with our colonies. At t=80min, we inoculated again our tube of Medium A.

- We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171

- Spectrophotometer : blank made with medium A

Time (min) 0 20 40 60 80 100
OD650 0.2393 0.2504 0.2663 0.2807 0.3184 0.3672
Book1 3816 image001.gif

- Continue the protocol, add DNA (ECE153, 166, 171)

- Plate on antibiotic plates

  • New glycerol stocks

- 8 tubes : spin cells, throw away the supernatant, add 500μL of glycerol 60%

- 8 tubes : spin cells, keep only 100μL of medium B, resuspend cells and add 500μL of glycerol 60%

- Put them in the freezer at -80°C

Dealing with R0040, R0062, S0168, I13522 for PA

  • Plated R0040, R0062, S0168 received from MIT onto Amp100 plates and incubate at 37°C
  • Pciked 4 single colonies for each after colonies have grown and incubate overnight

Check I13522 biobrick DNA

  • PCR biobrick DNA from 2007 wells
  • Run 0.8% EtBr E-gel
  • Lane 5: I13522
  • Lane 7: Hyperladder I

Result:

  • Size of band is too small under 1000bp should be 2375bp instead
  • Transform TOP10 again but with I13522 from 2008 wells

August 20th

Check vector ECE190

- Plasmid miniprep

- Nanodrop

- Double digest : 6μL of SDW, 8μL of DNA, 1μL of buffer, 1μL of XbaI and 1μL of PstI

- Run on a gel

Boodfg image001.gif

- Result : 2 bands (lane3, a little bit more than 3000b and a little bit more than 5000b) :ok!

August 21st

Check ECE188

- Plasmid miniprep

- Double digest with XbaI and PstI

- Gel (lane6)

Photop.gif

- Result : nothing! not enough DNA

August 22nd

Stock ECE112, 166, 153

- Plasmid miniprep (2 tubes of ECE112, 2 tubes of ECE166 and 1 tube of ECE153)

- Nanodrop

Transformation of Bacillus

  • For strain IA751 : transform with ECE166, 171, 153 and 112
  • For strain IA771 : transform with ECE166, 171
  • for glycerol stocks (one with 100μL of medium B and glycerol, and one with only glycerol) : transform with ECE166


- We used the same protocol than before with a few changes :

  • add sterile 50% glucose in medium A
  • don't keep samples after using them for spectrophotometer
  • spin each tubes and keep only 100μL of medium A (to concentrate cells) before adding DNA
Time (min) 0 20 40 60 80 100 120 130 140
OD650 for IA751 0.1817 0.1870 0.1929 0.2115 0.2543 0.3291 0.3695 0.4188 0.4224
OD650 for IA751 0.1818 0.1874 0.1907 0.2088 0.2452 0.3182 0.3575 0.4228 0.4314
Graph.gif

- For glycerol stocks, resuspend cells into 100μL of medium B, add DNA and wait for 30min, end plate them

August 23rd

Results of transformation from 23/08/2008

Vector Strain Antibiotic number of colonies Conclusion
ECE166 IA771, glycerol stock with medium B Cm5 one large colony and a lot of very small colonies Contamination?
Control IA771, glycerol stock with medium B Cm5 a lot of very small colonies Contamination?
ECE166 IA771, glycerol stock without medium B Cm5 many very small colonies Contamination?
Control IA771, glycerol stock without medium B Cm5 a few tiny colonies Contamination?
ECE166 IA751, fresh Cm5 9 large colonies, no small ones to test
Control IA751, fresh Cm5 no growth, scratches ok
ECE166 IA771, fresh Cm5 1 large colony, many small worm-like growth, scratches? to test
Control IA771, fresh Cm5 no colonies at all ok
ECE171 IA771, fresh Kan5 no colonies  ??
Control IA771, fresh Cm5 no colonies ok
ECE171 IA751, fresh Kan5 about 30 large colonies and 100 small colonies to test?
Control IA751, fresh Cm5 3 large colonies, about 20 small to medium colonies contamination?
ECE112 IA751, fresh Cm5 + Spc100 no growth  ??
Control IA751, fresh Cm5 + Spc100 no growth
ECE153 IA751, fresh Spc50 about 500 medium to large colonies to test
Control IA751, fresh Spc50 no growth ok

August 26th

Xylose test

  • for ECE153

- Prepare 4 tubes with 10mL LB and Spc50, inoculate each tube with a colony from IA751 + ECE153

Starch plate

- prepare some blank agar plates

- add 1g of starch into 100mL of soft agar, mix, boil

- put a thin layer of soft agar with starch

- plate with IA751, IA771, IA751+ECE153 and IA751 (control plate, to see if we mixed some plates)

- Incubate at 37°C

Ery plate

- Plate IA751, IA771, IA751 (control plate) and IA751+ECE153


August 27th

Xylose test

  • Preparation of xylose tube

- Add 1mL of culture from yesterday, 1mL of xylose (0.1g/mL) and 8mL of LB and 45μL of Spc (do that for the 4 different colonies)

- Incubate 5hours

- Observation with microscope : some green task, but they do not really match with bacillus cells, not conclusive!

Starch plate

- Add 5mL of iodine solution on starch plate, wait for 1min, throw the liquid

- Result

Strain added vector Observation Conclusion
IA771 none no clearance zones ok
IA751 none big clearance zones ok
IA751 ECE153 no clearance zones trnsformation worked!
IA751 or IA771 none or ECE171 big clearance zones we have some colonues on control plate with IA751, contamination? or antibiotic resistance?

Ery test

- IA771 alive and IA751 dead, confirmation that we did not mix plates (same conclusion than with starch plate, last line)

Test glycerol stocks of competent bacillus

- Pellet bacillus cells, add 100μL of prewarmed mediumB, add 0.6μg of DNA, incubate 30min at 37°C by shaking, plate

Strain added vector Antibiotic
IA771 none blank plate (to check they are alive)
IA771 none Cm5 (negative control)
IA771 ECE166 Cm5
IA751 none blank plate (to check they are alive)
IA751 none Cm5 (negative control)
IA751 none Spc50 (negative control)
IA751 ECE112 Cm5
IA751 ECE153 Spc50

August 28th

Results from Bacillus transformation with glycerol stocks

Strain added vector Antibiotic number of colonies Conclusion
IA771 none blank plate confluent colonies cells are alive
IA771 none Cm5 0 no contamination
IA771 ECE166 Cm5 2 transformation ok, very low efficiency
IA751 none blank plate confluent colonies cells are alive
IA751 none Cm5 0 no contamination
IA751 none Spc50 0 no contamination
IA751 ECE112 Cm5 0 problem, we can not manage to transform this vector
IA751 ECE153 Spc50 156 transformation ok, good efficiency


Test AmyE insertion

  • Colonies PCR for Bacillus (IA751 transformed with ECE153, IA751, and IA771)

- 20μL of lysozyme, a few colonies for each

- Cycle : 15min qt 37°C, 15min at 99°C, 1min at 4°C, 1min at 99°C and 1min at 4°C

- for each PCR : 12μL of cells, 1μL of cells, 1μL of primer1, 1μL of primer2, 5μL of master mix

- PCR (iGEM program)

- run a gel

Photoq.gif
  • Result : nothing on the gel!!! But we can not manage to PCR anything... there is a problem in our PCR protocol, or products!

August 29th

Stock of ECE112

- Bulk up ECE112 (Amp100) rfom 10mL LB (28/08), take 1mL and add to 100mL of LB in big flask

- Grow overnight in 37°C incubator with vigorous shaking

- Aliquot in 10mL tubes, pellet and freeze cell pellets

Transformation of glycerol stock

  • Strian : IA751

- Transfer into Eppendorf tubes and spin down for 30min

-Remove glycerol and add medium B

- Add ECE112 and ECE153, and one control tube

- incubate for 30min in 37°C

- Plate out ECE112 (Cm5), ECE153 (Spc50), DNA less (blank, Cm5, Spc50)

September 1st

Result for Bacillus transformation (27/08)

Strain Vector Antibiotic number of colonies Conclusion
IA751 None Spc50 0 ok
IA751 ECE153 Spc50 about 200 very good efficiency
IA751 None Blank confluent cells are alive
IA751 None Cm5 a lot problem of contamination (bacillus??? or E.coli???)
IA751 ECE112 Cm5 a lot to check

New Bacillus transformation (to make glycerol stocks)

  • 2 attempts with plates from 12/08 and 18/08, no growth... Cells may be dead.
  • New plate of IA771 and IA751 (with glycerol stocks)

September 2nd

Transformation of Bacillus

  • with fresh plates from yesterday, last protocol
Time (min) 0 20 40 60 85 105 125
OD650 for IA751 0.1192 0.1330 0.1736 0.2238 0.3124 0.4523 0.5113
OD650 for IA771 0.1144 0.1216 0.1593 0.2296 0.3476 0.4302 0.5565
Transf0209.gif
  • t0 (IA751) = 100min
  • t0 (IA771) = 110min
  • Transformation

- IA751 on Cm5 and Spc 50 (control DNA less plates)

- IA751 + ECE112 (1μg of DNA) on Cm5

- IA751 + ECE153 (0.6μg of DNA) on Scp50

- IA771 on Cm5 (control)

- IA771 + ECE166 on Cm5

  • Glycerol stocks (about 20 tubes of each)

Check ECE112 stock

- Double digest with XbaI and EcoRI

Gel2.gif

- Gel : problem only one band (single digest???)

September 3rd

Result from bacillus transformation with fresh competent cells (02/09)

strain antibiotic added vector observation conclusion
IA751 none none confluent colonies cells are alive
IA751 Spc50 none no colonies no contamination
IA751 Spc50 ECE153 70 colonies transformation to check, efficiency : about 115 colonies/μg of DNA
IA771 none none confluent colonies cells are alive
IA771 Cm5 none no colonies no contamination
IA771 Cm5 ECE166 16 colonies transformation to check, efficiency : about 27 colonies/μg of DNA
IA771 Cm5 ECE112 41 small colonies transformation to check, efficiency : about 41 colonies/μg of DNA

With 1μg of DNA, we manage to obtain colonies with ECE112. We will check our transformation with starch plates.

Check ECE112 stock

- Single and double digest with XbaI and EcoRI

Gel3.gif

- Gel

  • Lane 3 : HyperladderI
  • Lane 4 : ECE112, 100mL flask, single digest with XbaI (expected size about 10,000bp)
  • Lane 5 : ECE112, 100mL flask, single digest with EcoRI (expected size about 10,000bp)
  • Lane 6 : ECE112, 100mL flask, double digest (expected size about 3200bp and 6,900bp)
  • Lane 7 : ECE112, 10mL flask, single digest with XbaI (expected size about 10,000bp)
  • Lane 8 : ECE112, 10mL flask, single digest with EcoRI (expected size about 10,000bp)
  • Lane 9 : ECE112, 10mL flask, double digest (expected size about 3200bp and 6,900bp)
  • Lane 10 : HyperladderI

- Results : ok

Test glycerol stocks (from 02/09) of competent bacillus

strain antibiotic added vector quantity of DNA
IA751 none none 0
IA751 Spc50 none 0
IA751 Spc50 ECE153 0.6
IA771 none none 0
IA771 Cm5 none 0
IA771 Cm5 ECE166 0.6

Starch plates

- Use blank plates

- Melt 100mL of Soft Agar, and add slowly 1g of starch

- Mix, boil to sterilize

- Pour on blank plate

- Put 4 or 5 colonies on each plate

  • IA751 from blank plate (positive control)
  • IA771 from blank plate (negative control)
  • IA751 transformed with ECE112 (Cm5, 29/08)
  • IA751 control (Cm5, 29/08)
  • IA751 + ECE112 (Cm5, 02/09)
  • IA751 + ECE153 (Cm5, 02/09)
  • IA751 + ECE153 (Cm5, 29/08)


September 4th

Result of Bacillus transformation (glycerol stock of competent cells)

strain antibiotic added vector observation conclusion
IA751 none none confluent colonies cells alive
IA751 Spc50 none nothing no contamination
IA751 Spc50 ECE153 15 ok, efficiency 25 colonies/μg of DNA
IA771 none none confluent colonies cells alive
IA771 Cm5 none nothing no contamination
IA771 Cm5 ECE166 15 ok, efficiency 5 colonies/μg of DNA

We have no contamination in our glycerol stocks. However, the efficiency of glycerol stocks seems to be lower than these of fresh competent cells.

Result of starch plates

We had 5mL of iodine solution, wait for 1min and observe.

Controls (the small zone of clearing are contamination, we have not yet manage to find how to avoid them)

Negative control : IA771, no zone of clearing

Photoesfr.gif

Positive control : IA751, big zones of clearing

Photoesr.gif
strain vector photo observation conclusion
IA751 none
Photoer.gif
 big zones of clearing for each colony ok
IA771 none
Photo2.gif
 no zone of clearing for each colony ok
IA751 ECE112/none (29/08)
Photo3.gif
 clearing zones for control, so no trnasformation, it is maybe E.coli, 1 clearing zone out of 3 colonies, it is certainly contamination, but that means that some colonies are transformed ok
IA751 ECE112 (02/09)
Photo4.gif
 1 zone of clearing out of 4 colonies some colonies are not transformed and have a resistance to Cm5 (contamination?)
IA751 ECE153 (28/08)
Photo5.gif
 no zone of clearing 5 transformed colonies
IA751 ECE153 (02/09)
Photo6.gif
 2 zone of clearing out of 5 colonies some colonies are not transformed and have a resistance to Spc50 (contamination?)

Colony PCR from bacillus subtilis colonies

We want to check the size of the insert in the AmyE region(IA751) after transformation with an integration vector (to check if we have a single cross over or not).

We are going to test our transformation of ECE112 and 153.

We already tried this protocol, but our PCR did not work, so we tried again, with one sample of chromosomal DNA from bacillus to check if our transformation works.

- add 20μL of 0.05mg/mL lysozyme into a PCR tube

- add a few colonies into this tube until you obtain a dense solution. We test :

  • IA751
  • IA751 + ECE153 (29/08, 02/09, 03/09)
  • IA751 + ECE112 (02/09)

- 15min in 37°C, then 15min in 99°C, 1min in 4°C, 1min in 99°C, 1min in 4°C

- For IA751, IA751 + ECE112, IA751 + ECE153, and bacillus chromosomal DNA, add 12μL of SDW, 5μL of MM, 1+1μL of primers (amyE primers), 1μL of the solution prepared befor with cells (or DNA)

- PCR

- Gel

  • Lane1 : hyperladderI
  • Lane2 : IA751 + ECE153 (29/08)
  • Lane3 : IA751 + ECE153 (02/09)
  • Lane4 : IA751 + ECE153 (03/09)
  • Lane5 : IA751 + ECE112 (02/09)
  • Lane6 : IA751
  • Lane7 : Chromosomal DNA
Photoed.gif

- Result : only chromosomal DNA worked. That means that our primers are ok, but we have to find a protocol for chromosomal DNA miniprep of bacillus to be able to PCR something into the genome of bacillus.

  • New attempt without the lyse process (only protocol of single colony PCR, like for E.coli)

- Gel

  • Lane1 : hyperladderI
  • Lane2 : IA751 + ECE153 (29/08)
  • Lane3 : IA751 + ECE153 (02/09)
  • Lane4 : IA751 + ECE153 (03/09)
  • Lane5 : IA751 + ECE112 (02/09)
  • Lane6 : IA751
  • Lane7 : Chromosomal DNA
Photoefr.gif

- Result : Lane4, we have something but too small.


September 5th

Starch plates

Prepare some starch plates. We want to test our transformation but we can not manage to PCR Bacillus. So we are going to try to extract chromosomal DNA from Bacillus. We make these starch plates to test it with colonies which have positive control with amylase test.

- Prepare starch plates (you can find the protocol here)

- Plate 4 different plates :

  • 2 colonies of IA75 (positive control) and 3 colonies of IA771 (negative control)
  • 5 colonies of IA751 transformed with ECE153 from 29/08
  • 3 colonies of IA751 transformed with ECE153 from 02/09 and 3 colonies from 03/09
  • 5 colonies of IA751 transformed with ECE112 from 02/09


September 6th

Results of starch plates

We had 5mL of iodine solution, wait for 1min and observe.

strain vector photo observation conclusion
IA751 none
100px
 big zones of clearing for IA751 (right), no zone of clearing for IA771 (left) IA751 : positive control and IA771 : negative control
IA751 ECE153 from 02/09 (on left)
100px
 colony 1 : zone of clearing, no transformation; colony 2 and 3 : no zone of clearing, transformation ok 2 transformed colonies out of 3
IA751 ECE153 from 03/09 (on right)
100px
 colony 1,2 and 3 : no zone of clearing, transformation ok transformation ok
IA751 ECE153 (28/08)
100px
 no zone of clearing for colonies 1, 2 and 3, impossible to say for colonies 4 and 5 because big contamination (so big zone of clearing) colony 1,2,3 : ok, no result for the others
IA751 ECE112 (02/09)
100px
 no zones of clearing transformation ok


September 8th

Prepare chromosomal DNA extraction from Bacillus

- Grow 2 tubes into LB for

  • IA751
  • IA751 + ECE153 from 03/09 (for tissue kit)
  • IA751 + ECE153 from 02/09
  • IA751 + ECE112 from 05/09

PCR AmyE from ECE112

- Add 12μL of SDW, 5 μL of MM, 1μL + 1μL of each primers (for AmyE back and AmyE front) and 1μL of ECE112

  • Lane1 : HyperladderI
  • Lane4 : AmyE front
  • Lane5 : AmyE front
  • Lane6 : AmyE back
  • Lane7 : AmyE back
Gelsdgsdg.gif
  • Results : good band for AmyE front, very low band for AmyE back


September 9th

Chromosomal DNA extraction

- ZR soil microbe DNA kit for

  • IA751
  • IA751+ECE112
  • IA751+ECE153

- Nanodrop

product (chromosomal DNA) concentration (ng/μL)
IA751 75
IA751 + ECE112 48.6
IA751 +ECE153 74.2


- PCR with AmyE detect primers (12μL of SDW, 5μL of MM, 1+1μL of each primer, 1μL of chromosomal DNA or plasmid)

- Gel

  • Lane1 : HyperladderI
  • Lane2 : AmyE Back
  • Lane3 : AmyE Front
  • Lane4 : Bacillus chromosomal DNA (given by Duncan to check)
  • Lane5 : ECE112 (plasmid)
  • Lane6 : ECE153 (plasmid)
  • Lane7 : chromosomal DNA IA751 + ECE112
  • Lane8 : chromosomal DNA IA751 + ECE153
  • Lane9 : chromosomal DNA IA751
Photosdfscontrols.gif
  • Results

- Only something for IA751, and ECE153 (pure plasmid)???

- New PCR with 4μL of chromosomal DNA and new PCR program

- New gel

  • Lane1 : IA751 (chromosomal DNA)
  • Lane2 : ECE153 (chromosomal DNA)
  • Lane3 : ECE112 (chromosomal DNA)
  • Lane4 : ECE112 (plasmid)
  • Lane5 : ECE153 (plasmid)
  • Lane6 : Bacillus chromosomal DNA (given by Duncan)
  • Lane7 : HyperladderI
  • Lane8 : agrA
  • Lane9 : agrB
  • Result
Photos09092.gif

- No insert in IA751 transformed with ECE153 and ECE112 (same size than control!) and however the amylase test is positive for this colony....

PCR AmyE again

- 24μL of SDW, 10μL of MM, 2μL of each primers, 2μL of ECE112

  • Results (cf gel, lane 2 and 3)

- Very good result for amyE front, nothing for AmyE back

September 10th

Check PCR for pSBINT1 vector

- Gel

  • Lane2 : HyperladderI
  • Lane3 : rep+bla
  • Lane4 : PCR of I714062
  • Lane5 : PCD of J04630
  • Result
Gel11093.gif

- One band for rep, but not very clear, we should try again

- for the other parts, we have a good band but the wrong size, these biobricks seem to be bad, we will order them from the MIT

September 11th

PCR for pBSINT1 biobrick vector

  • PCR rep+bla part from I732007 (pSB1A3 backbone)

- Add 12μL of SDW, 5μL of MM, 1μL of rep fwd primer, 1μL of rep rev primer and 1μL of I732007 (program iGEM new)

  • PCR I714062 and J04630 with VF and VR. We want to extract the cutting sites of the biobrick vector with GFP inside (in the future we would prefer to make our biobrick vector with a RSB for B.S. inside)

- Add 9μL of SDW, 5μL of MM, 1μL of VF, 1μL VR and 4μL of each biobrick (program iGEM new)

x

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