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Revision as of 17:56, 28 October 2008

August, 16th

Agr A and C ligation to pSB4C5

Ligation was performed using Fermentas Rapid Ligation kit according to their protocol.

After ligation, samples were visualized on a gel, but little product of the correct size was seen.

For agrA we should have gotten a band of size 3700 (3000plasmid + 700 insert)

For agrC we should have gotten a band of size 4300 (3000plasmid + 1300insert)

The bands of appropriate sizes were extracted for transformation.

August, 18th

Check promoters

On Friday, PCR out promoters, we want to check them.

- Run a gel

- Results : good size of bands!!!

Transformation of AgrA and AgrC

AgrA and C gell-extracted ligation was transformed into Top 10 cells using standard protocol. Puc9 was used as a positive control.

August, 19th

Results of AgrA and C transformation

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.

Lux parts

To make the Lux Receiver, we need 4 different parts ;

R0040, TetR repressible promoter

SO168, luxR + double terminator

R0062, promoter activated by luxR

JO4630 (GFP + double terminator)

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. 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.

- Plate on antibiotic plates and do LB stocks of single colony from the MIT stock (R0040, R0062 and S0168).

- Put on Kan plates 4 different colonies from J04630 (transformation Amp plate) and also incubate these colonies into LB

August, 20th

Check Lux components

- Single colony PCR for :

R0040 (MIT stuff)

R0062 (MIT stuff)

4 different colonies of S0168 (from a transformation plate from 12/08)

4 different colonies of J04630 (from a transformation plate from 12/08)

- 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

Gel 1

Lane2 : Hyperladder1
Lane3 : JO4630, colony 1
Lane4 : JO4630, colony 2
Lane5 : JO4630, colony 3
Lane6 : JO4630, colony 4
Lane7 : HyperladderI

Gel 2

Lane2 : HyperladderI
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

300px

- Results

R0040 and R0062 : one big band of about 300b (expected size 293), OK!

S0168 : one band of about 400b for the 4 different colonies (expected size 1234!), bad! This plate does not contain S0168

J04630 (colonies 2 and 4) : one band of about 1100b (expected size 1173), OK!

J04630 (colony 1) : one good band plus another band...

J04630 (colony 3) : one band of about 600b, bad!

Ligation

- Materials :

AgrA
AgrB
AgrC
AgrD
Pupp
Pspac
Ppac
Pxyl
RBS S
RBS W
psB4C5

- Double digest of PCR products

- Run vector, AgrA and AgrD on a gel

- DNA clean and concentrator for AgrA, B,C and D, promoters

- Microclean for both RBS

- Nanodrop

	260/280	ng/μL
AgrA	1.66	16.4
AgrB	1.91	23.5
AgrC	1.99	35.9
AgrD	2.13	4.9
Pxyl	1.54	5.6
Ppac	1.49	4.6
Pspc	1.62	9.6
Pupp	1.88	8.5
RBS S	2.44	29
RBS W	1.44	10.7

- Extract plasmid annd Agr from gel and clean

- Ligation

August, 21st

Transformation of ligation products

- Spin chemically competent TOP10, add 100μL of CaCl2 solution

- Add 5μL of DNA (ligation products), and 1μL of PUC9

- Continue the protocol of transformation

J04630

- Plate good colonies from yesterday on Kan25 and put in LB (for plasmid stock for tomorrow)

Plasmid stocks

- Plasmid miniprep R0040 and R0062

August, 22nd

New PCR of promoters

August, 23rd

Check promoters (after PCR from bacillus vectors)

- Gel

Lane1 : Hyperladder 5
Lane2 : Pxyl
Lane3 : Pspac
lane4 : Ppac
Lane5 : Pupp

Size is ok.

August, 27th

New transformation of ligation products

Products : agrD, Pupp, Pspac, RBS W, RBS S (from ligation with biolabs kit)

- Competent top 10 from the freezer

- Add 5μL of ligation products, and 1.2μL of PUC9

August, 28th

Results from transformation of ligation products

- nothing on plate, even on control plate

Reasons ?

- cells non competent anymore (try with fresh competent cells)

- not enough DNA (try with 10μL of DNA)

- very short ligation products

Transformation of ligation products (new)

Products to ligate : Pupp, Pspac, agrD, RBS S and RBS W

- new fresh competent TOP 10

- 5μL of DNA (1.5μL of PUC9)

- 2h30 in the incubator

August, 29th

Results of transformation with our ligation products

Everything grew! Better efficiency with electrop. than with chemical protocol

Single colony PCR to check our transformation

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)
Pupp	2	2	0
Pspac	2	2	0
RBS S	3	0	2
RBS W	3	0	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)

In the death plasmid, the VF-VR size is about 280b.

Transformed products	size of the product (with cutting sites)	expected size after PCR (about)
Pupp	255	480
Pspac	125	350
RBS S	56	280
RBS W	56	280
agrD	200	430

Result : nothing, even no ladder, problem with the gel!

- run again on a e-gel

Lane1 :ladder 100bp
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

Result : nothing, just the primers! Problem with our PCR

Plate biobricks from MIT

E0840
B0014
I712007
C0012
B0015
C0061
R0063

September, 2nd

Check transformation in E.coli of our ligated products

- Single colony PCR with VF and VR : add 10μL of SDW, 5μL of MM, 1μL of VF, 1μL of VR and 3μL of cells

inserted products	number of colonies to check (chemical transf.)	number of colonies to check (electrop. transf.)
RBS S	3	2
RBS W	3	2
Pspac	2	2
Pupp	2	2
agrD	3	2

- Gel (3% of agarose)

Result

gel	lane	inserted product (name of the colony)	observation	conclusion
top	3	RBS S (1)	one band (260bp)	size of the insert, RBS too small to see on a gel
top	4	RBS S (2)	one band (260bp)	size of the insert, RBS too small to see on a gel
top	5	RBS S (4)	nothing	?
top	6	RBS S (5)	nothing	?
top	7	RBS W (1)	2 band (260bp + 350bp)	size of the insert, RBS too small to see on a gel + something else?
top	8	RBS W (2)	one band (350bp)	too big
top	9	RBS W (4)	one band (260bp)	size of the insert, RBS too small to see on a gel
top	10	RBS W (5)	one band (260bp)	size of the insert, RBS too small to see on a gel
top	11	Pspac (1)	2 band (260bp + 350bp)	maybe problem with products loaded on gel (exactly the same bands than for RBS W)
top	12	Pspac (2)	one band (450bp)	size of Pupp?
top	13	Pspac (3)	one band (450bp)	size of Pupp?
bottom	3	Pspac (4)	nothing	?
bottom	4	Pupp (1)	one band (400bp)	size of Pspac?
bottom	5	Pupp (2)	one band (400bp)	size of Pspac?
bottom	6	Pupp (3)	one band (slightly lower than 400bp)	size of Pspac?
bottom	7	Pupp (4)	one band (400bp)	size of Pspac?
bottom	8	agrD (1)	2 bands (380 and 450bp)	big band is ok
bottom	9	agrD (2)	2 bands (380 and 450bp)	big band is ok
bottom	10	agrD (3)	no bands	?
bottom	11	agrD (4)	strong band (450bp)	ok
bottom	12	P2	one band (350bp)	ok

September, 3rd

Checking the insert of promoters

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).

We also want to PCR again all promoters (Pspac, Pxyl, Pupp and Ppac) from plasmids to have more.

Picture of the gel with the different promoters

promoter	colony	primers	name
Pspac	1	Pspac primers	A
Pspac	1	Pupp primers	B
Pspac	2	Pspac primers	C
Pspac	2	Pupp primers	D
Pupp	1	Pupp primers	E
Pupp	1	Pspac primers	F
Pupp	3	Pupp primers	G
Pupp	3	Pspac primers	H

- Single colony PCR : add 10μL of SDW, 5μL of MM, 1μL + 1μL of primers, 3μL of cells