Team:Groningen/Notebook/epse overexpression
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epsE overexpression
epsE is an inhibitor of motility and produces glycosyltransferase required for EPS biosynthesis. Overexpressing it should increase biofilm growth.
To overexpress epsE in Bacillus subtilus.
The transformation was unsuccessfull.
None
00:00, 16 Juli 2015 - 00:00, 16 Juli 2015
A plasmid was created with RBS in front of epsE. The plasmid was transformed into E. coli.
Restriction
Cup
Contents
1
20 µL plasmid that includes RBS, 1 µL EcoRI, 1 µl SpeI, 5µL NEB cutsmart and 23 µL demiwater
2
20 µL plasmid that includes epsE, 1 µL XbaI, 1 µL pstI, 5µL NEB 2.1 and 23 µL demiwater
3
10 µL kanamycin backbone (pSB1K3.m1), 1 µL EcoRI, 1 µL pstI, 5 µL NEB 2.1 and 33 µL demiwater
Contents of cups.
The cups were incubated for 1 hour at 37 °C. Then a Thermo Scientific purification kit was used on each cup.
Ligation
Competent cells (NEB5-alpha competent E. coli high efficiency) were defrosted. 10 µL of competent cells were pipetted in cup A and the cup was stirred using a pipet point. The cup was then put on ice. After leaving it on ice for 15 minutes the mixture was incubated at 42 °C for 15 minutes. Subsequently, the mixture was placed on ice for 2 minutes. 900 µL of LB medium was added to the mixture and the cup was centrifuged at 8000 rpm for 2 minutes. The supernatant was removed by pipetting and the pellet was resuspended with fresh medium. Finally, the mixture was put on LB kanamycin plates and left in the stove at 37 °C during the night.
The RBS was smaller than allowed by the DNA purification kit specifications, but colony pcr showed that this did not cause any problems.
00:00, 21 Juli 2015 - 00:00, 21 Juli 2015
To overexpress epsE in B. subtilus we place a promotor (which constantly promotes the transcription of epsE) accompanied by a RBS, in a plasmid.
Pveg, RBS + epsE in pSB1A3
Cup
Contents
4
20 µL plasmid that includes RBS + epsE (acquired with miniprep, obtaining plasmide DNA), 1 µL XbaI, 1 µL pstI, 5µL NEB 2.1 and 23 µL demiwater
5
20 µL Pveg (number 2 in Marieke’s boekje), 1µL EcoRI, 1µl SpeI, 5µL NEB cutsmart and 23 µL demiwater
6
10 µL ampicillin backbone (pSB1A3), 1 µL EcoRI, 1 µL pstI, 5 µL NEB 2.1 and 33 µL demiwater
Contents of cups.
Samples were incubated for 1 hour @ 37 °C, and purified using PCR.
Ligation
In cup A 7 µL out of cup 4, 7 µL out of cup 5 and 6 µL out of cup (????), 2 µL 10x T4 buffer, 0.5 µL T4 ligase enzyme.
The mix was left to rest for 1 hour at room temperature
Cup
Contents
A
7 µL out of cup 4, 7 µL out of cup 5 and 6 µL out of cup (????), 2 µL 10x T4 buffer, 0.5 µL T4 ligase enzyme.
Ligation mix
The mix was left to rest for 1 hour at room temperature.
Transformation
Competent cells (NEB5-alpha competent E. coli high efficiency) were defrosted. 10 µL of competent cells were pipetted in cup A and the cup was stirred using a pipet point. The cup was put on ice. After leaving it on ice for 15 minutes the mixture was incubated at 42 °C for 15 minutes. Subsequently, the mixture was placed on ice for 2 minutes. 900 µL of LB medium was added to the mixture and the cup was centrifuged at 8000 rpm for 2 minutes. The supernatant was removed by pipetting and the pellet was resuspended with fresh medium. Finally, the mixture was put on LB ampicillin plates and left in the stove at 37 °C during the night.
LB amp plates were accidently left in the refrigerator instead of being placed at 37 ° C one hour in advance.
Therefore the competent cells were resuspended in suspernatant after centrifugation and placed in a 37 ° C water bath for one extra hour while the LB amp plates were at 37 ° C.
00:00, 22 Juli 2015 - 00:00, 22 Juli 2015
colony pcr
Pcr was performed to see if overexpression of epsE in B. subtilus was successful.
8 Eppendorf tubes were filled with 30 µL of demiwater each. An equal number of colonies were picked from the plate and suspended in the Eppendorf tubes. Then 8 pcr cups were filled with 48 µL mastermix and 2 µL of suspended bacteria each.
PCR results
?
placed on 1% agarose gel.
Band was not as high as expected, pveg promotor was missing.
00:00, Juli 27 2015 - 00:00, Juli 27 2015
A plasmid was created with RBS in front of epsE. The plasmid was transformed into E. coli.
RBS and epsE
Cup
Contents
1
5 µL plasmid that includes RBS, 1µL EcoRI, 1µl SpeI, 2µL NEB 2.1 and 12 µL demiwater
2
10 µL plasmid that includes epsE, 1 µL XbaI, 1 µL pstI, 2µL NEB 2.1 2.1 and 7 µL demiwater
Contents of cups used.
Cups were incubated at 37 °C for one hour. Then cup 1 was placed on ice and the contents of cup 2 were loaded on 1% agarose gel, which was then ran at 100v. The band height was checked after one hour and found to be around 3000 bps as expected. The band was cut from the gel and purified using a Thermo Scientific DNA purification kit.
In contrast to the protocol binding buffer was added to the gel in a 1:2 ratio. The sample was vortexed until the gel diluted in the buffer.
Ligation
Cup
Contents
A
4 µL of purified epsE, 8 µL of unpurified RBS, 2 µL 10x T4 buffer and 1 µL T4 ligase enzyme
Rested O/N at 4 °C
Ligation mix
Transformation
Competent cells (NEB5-alpha competent E. coli high efficiency) were defrosted. 10 µL of competent cells were pipetted in cup A and mixed using a pipet point. The cup was put on ice. After leaving it on ice for 15 minutes the mixture was incubated at 42 °C for 15 minutes. Subsequently, the mixture was placed on ice for 2 minutes. 900 µL of LB medium was added to the mixture and the cup was centrifuged at 8000 rpm for 2 minutes. The supernatant was removed by pipetting and the pellet was resuspended with fresh medium. Finally, the mixture was put on LB ampicillin plates and left in the stove at 37 °C during the night.
The plates showed no colonies.
00:00, 29 Juli 2015 - 00:00, 29 Juli 2015
with previous restrictions RBS would be placed in front of epsE, therefore the cloning plan was adapted.
Cup
Contents
2
10 µL of plasmids that including promoters, 1µL EcoRI, 1µl SpeI, 2µL NEB 2.1 and 7 µL demiwater
3
10 µL plasmids including promoters, 1µL EcoRI, 1µl SpeI, 2µL NEB 2.1 and 7 µL demiwater
Plasmid cups.
Cups were incubated at 37 °C for one hour. The DNA was then purified using a Thermo Scientific DNA purification kit. The amount of DNA was measured using a Nanodrop spectrophotometer (see table below).
epsE
#6
#7
4 ng/µL
5.5 ng/µL
2.5 ng/µL
DNA concentrations.
Ligation
Cup
Contents
2+2
5 µL of purified epsE, 10 µL of #6, 2 µL 10x T4 buffer and 1 µL T4 ligase enzyme
2+2
5 µL of purified epsE, 10 µL of #6, 2 µL 10x T4 buffer and 1 µL T4 ligase enzyme
Ligation cups
The cups were left to rest at room temperature for one hour.
Transformation
Competent cells (NEB5-alpha competent E. coli high efficiency) were defrosted. 10 µL of cup 2+2 and cup 2+3 were mixed with 10 µL of competent cells. The mix was stirred using a pipet point and subsequently put on ice. After 15 minutes on ice the mixture was incubated at 42 °C for 15 minutes. The mixture was placed on ice for 2 minutes. 900 µL of LB medium was added to the mixture and the cup was centrifuged at 8000 rpm for 2 minutes. The supernatant was removed by pipetting and the pellet was resuspended with fresh medium. Finally, the mixture was put on dried LB ampicillin plates and left in the stove at 37 °C during the night.
Several colonies were found. The promoters were in place in front of epsE. Unfortunately, the RBS was missing.
00:00, 30 Juli 2015 - 00:00, 30 Juli 2015
Before inserting promoters, the size of the miniprepped epsE plasmid was checked.
Cup 1 was filled with 10 µL ligated epse, RBS and kanamycin, 1µL EcoRI, 1µl XBAI, 2µL NEB 2.1 and 7 µL demiwater. This was left to rest for two hours in a 37 °C shaking bath. The contents of the cup were then placed on a 1% gel after being diluted with 4 µL of 6x loading buffer. The gel was ran at 100V for 30 minutes. No bands were seen.
00:00, 31 Juli 2015 - 00:00, 31 Juli 2015
Before inserting promoters, the size of the miniprepped epsE plasmid was checked.
Cup 1 was filled with 10 µL ligated epse, RBS and kanamycin, 1µL EcoRI, 1µl XBAI, 2µL NEB 2.1 and 7 µL demiwater. This was left to rest for two hours in a 37 °C shaking bath. The contents of the cup were then placed on a 1% gel after being diluted with 4 µL of 6x loading buffer. The gel was ran at 100V for 30 minutes. No bands were detected. Likely cause is that the sample was not miniprepped.
00:00, 3 August 2015 - 00:00, 3 August 2015
Before inserting promoters, the size of the miniprepped epsE plasmid was checked.
Cup 1 was filled with 10 µL ligated epse, RBS and kanamycin, 1µL EcoRI, 1µl XBAI, 2µL NEB 2.1 and 7 µL demiwater. This was left to rest for two hours in a 37 °C shaking bath. The contents of the cup were then placed on a 1% gel after being diluted with 4 µL of 6x loading buffer. The gel was ran at 100V for 30 minutes. A band at 4000 bp was seen. The band was cut from the gel and purified using a Thermo Scientific purification kit at a 2:1 buffer/DNA ratio. The concentration was measured to be 1 ng/µL using a Nanodrop spectrophotometer.
Ligation