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Revision as of 22:12, 28 October 2008 (view source) Lboettger (Talk | contribs) (→September 3rd) ← Older edit		Revision as of 22:23, 28 October 2008 (view source) Lboettger (Talk | contribs) (→September 4th) Newer edit →
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	- Result : Lane4, we have something but too small.		- 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
-	<!-- ## Do not edit below this line unless you know what you are doing. ## -->
	__NOTOC__		__NOTOC__

---

Revision as of 22:23, 28 October 2008

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

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

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

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

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

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

- 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

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

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

• 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

- 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

- 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

- 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)

- 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

- 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

• 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

• 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

- 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

- 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

Positive control : IA751, big zones of clearing

strain	vector	photo	observation	conclusion
IA751	none		big zones of clearing for each colony	ok
IA771	none		no zone of clearing for each colony	ok
IA751	ECE112/none (29/08)		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)		1 zone of clearing out of 4 colonies	some colonies are not transformed and have a resistance to Cm5 (contamination?)
IA751	ECE153 (28/08)		no zone of clearing	5 transformed colonies
IA751	ECE153 (02/09)		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

- 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

- 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

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