Team:NKU China/Notebook

50μL PCR system X2
2X Taq Master Mix 25μL
C2-F 2μL
C2-R 2μL
p-C2 2μL
ddH2O 19μL
Total 50μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
58℃ 15 sec 30 cycles
72℃ 30 sec
72℃ 10 min
16℃ ∞
20μL ligation system
10X DNA Ligase Buffer 2μL
T4 DNA Ligase 1μL
pMD19 T-Simple Vector 1μL
C2-luxS 4μL
ddH2O 12μL
Total 20μL
Reaction condition: 16℃ overnight
20μL digestion system
10X FastDigest Buffer 2μL
BamH Ⅰ 1μL
T-lsrACDB 1μL
ddH2O 7μL
Total 20μL
Reaction condition: 37℃ for 40 min
100μL methylation system
10X BamH Ⅰ methyltransferase Buffer 10μL
BamH Ⅰ methyltransferase 1μL
S-adenosylmethionine 0.5μL
pWH-C2-luxS 80μL
ddH2O 8.5μL
Total 100μL
Reaction condition: 37℃ for 1 hour
Groups divided in this experiment
GR286 wild strain as control group
GR286ΔluxS GR286 without luxS gene
pWH-luxS luxS overexpression plasmid in GR286; without induced by xylose
pWH-luxS + xyl luxS overexpression plasmid in GR286; induced by xylose
pWH1520 empty plasmid in GR286 as control group
pHT-lsrACDB lsrACDB overexpression plasmid in GR286ΔluxS
pHT-01 empty plasmid in GR286ΔluxS as control group
Selecting positive clones by colony PCR
(No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-C2-luxS into GR286)

Notebook

Week1

In order to make sure our "consumer" efficient, we should first knock out the luxS gene in our engineering bacteria GR286(a simplified strain of Bacillus amyloliquefaciens LL3). We used a markerless gene replacement method to knock out the luxS gene.

Construction of targeting vector : the upstream and downstream of luxS gene were combined by over-lapping PCR and ligated into plasmid pKSU.

Transformed pKSU-ΔluxS into GR286, and selected out positive clones.

20μL PCR system
2X Taq Master Mix 10μL
pKSU-F 1μL
pKSU-R 1μL
Bacterium solution 1μL
ddH2O 7μL
Total 20μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
58℃ 30 sec 30 cycles
72℃ 1 min 30 sec
72℃ 10 min
16℃ ∞
Selecting positive clones by PCR

The transformants were cultured at 42℃ with chloramphenicol to select single-crossover clones.

20μL PCR system
2X Taq Master Mix 10μL
luxS-up-F 1μL
luxS-dn-R 1μL
Bacterium solution 1μL
ddH2O 7μL
Total 20μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
56℃ 30 sec 30 cycles
72℃ 2 min
72℃ 10 min
16℃ ∞
Selecting single-crossover clones using PCR
(No.1-4 are single-crossover strains, No.5 is positive control.)

Week2

The single-crossover strains were then cultured at LB medium and passaged every 12 hours for 4 generation.

Cultured the last generation at medium with 5-fluorouracil to select double-crossover clones. Regretfully, we didn't get the double-crossover clones.

20μL PCR system
2X Taq Master Mix 10μL
luxS-up-F 1μL
luxS-dn-R 1μL
Bacterium solution 1μL
ddH2O 7μL
Total 20μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
56℃ 30 sec 30 cycles
72℃ 2 min
72℃ 10 min
16℃ ∞
Selecting double-crossover clones using PCR
(No.1-5 are experimental groups, No.6 is wild GR286.The result showed that we failed to get the double-crossover clones.)

Week3

We cultured transformants at 42℃ with chloramphenicol again and selected the single-crossover clones successfully.

20μL PCR system
2X Taq Master Mix 10μL
luxS-up-F 1μL
luxS-dn-R 1μL
Bacterium solution 1μL
ddH2O 7μL
Total 20μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
56℃ 30 sec 30 cycles
72℃ 2 min
72℃ 10 min
16℃ ∞
Selecting single-crossover clones using PCR
(No.1&2&4 are single-crossover strains,No.5 is positive control. )

The single-crossover strains were then cultured at LB medium and passaged every 12 hours for 4 generations.

Cultured the last generation at medium with 5-fluorouracil to select double-crossover clones. We finally got our aimed strain—GR286ΔluxS.

20μL PCR system
2X Taq Master Mix 10μL
luxS-up-F 1μL
luxS-dn-R 1μL
Bacterium solution 1μL
ddH2O 7μL
Total 20μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
56℃ 30 sec 30 cycles
72℃ 2 min
72℃ 10 min
16℃ ∞
Selecting the strain lacking of luxS gene using PCR
(The No.4 is the aimed strainGR286ΔluxS)

Week4

Cultured the GR286ΔluxS strain and made it competence for future use.

Cloned the lsrACDB gene from Bacillus thuringiensis and ligated it to T-vector.

50μL PCR system X2
2X Taq Master Mix 25μL
lsrACDB-F 2μL
lsrACDB-R 2μL
Bacterium solution 2μL
ddH2O 19μL
Total 50μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
57℃ 30 sec 30 cycles
72℃ 4 min 30 sec
72℃ 10 min
16℃ ∞
20μL ligation system
10X DNA Ligase Buffer 2μL
T4 DNA Ligase 1μL
pMD19 T-Simple Vector 1μL
lsrACDB 3μL
ddH2O 13μL
Total 20μL
Reaction condition: 16℃ overnight

Transformed the T-lsrACDB into DH5α and coated plate, and then selected positive clones by colony PCR.

20μL PCR system
2X Taq Master Mix 10μL
M13F 1μL
M13R 1μL
Bacterium solution 1μL
ddH2O 7μL
Total 20μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
59℃ 30 sec 30 cycles
72℃ 4 min 30 sec
72℃ 10 min
16℃ ∞
Selecting positive clones by PCR
(No. 3&4 are positive results)

After restriction enzyme digestion verification, we sent them to sequencing. Unfortunately, the sequencing result showed some mutations in cloning gene.

20μL digestion system
10X FastDigest Buffer 2μL
BamH Ⅰ 1μL
T-lsrACDB 1μL
ddH2O 7μL
Total 20μL
Reaction condition: 37℃ for 40 min
Restriction enzyme digestion verification
(No.1 are lsrACDB fragement, No.2 are linearized T-vector.)

We repeated the process of gene cloning but there were still some mutations.

We finally decided to request the gene company to synthesize the lsrACDB gene.

Week5

This week, we started to construct another controller―supplier.

We cloned a strong promoter C2 from former kit and cloned luxS gene from GR286.

50μL PCR system X2
2X Taq Master Mix 25μL
C2-F 2μL
C2-R 2μL
p-C2 2μL
ddH2O 19μL
Total 50μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
58℃ 15 sec 30 cycles
72℃ 30 sec
72℃ 10 min
16℃ ∞
50μL PCR system X2
2X Taq Master Mix 25μL
luxS-F 2μL
luxS-R 2μL
Bacterium solution 1μL
GR286 2μL
ddH2O 19μL
Total 50μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
59℃ 30 sec 30 cycles
72℃ 30 sec
72℃ 10 min
16℃ ∞
Cloning promoter C2 and luxS using PCR
(NO.1&2 are C2, No.3&4 are luxS)

Fuse the two segments together by fusion PCR, and ligated it into T-vector. Then, transformed the vector into DH5α.

50μL PCR system X2
2X Taq Master Mix 25μL
C2-F 2μL
luxS-R 2μL
C2 2μL
luxS 2μL
ddH2O 17μL
Total 50μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
59℃ 30 sec 30 cycles
72℃ 40 sec
72℃ 10 min
16℃ ∞
20μL ligation system
10X DNA Ligase Buffer 2μL
T4 DNA Ligase 1μL
pMD19 T-Simple Vector 1μL
C2-luxS 4μL
ddH2O 12μL
Total 20μL
Reaction condition: 16℃ overnight

Selected the positive clones by colony PCR.

20μL PCR system
2X Taq Master Mix 10μL
M13-F 1μL
M13-R 1μL
Bacterium solution 1μL
ddH2O 7μL
Total 20μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
59℃ 30 sec 30 cycles
72℃ 40 sec
72℃ 10 min
16℃ ∞
Selecting positive clones by colony PCR

We chose 4 positive strains to culture overnight and extracted the plasmid. After restriction enzyme digestion verification, we sent them to sequencing.

20μL digestion system
10X FastDigest Buffer 2μL
BamH Ⅰ 1μL
T-lsrACDB 1μL
ddH2O 7μL
Total 20μL
Reaction condition: 37℃ for 40 min
Restriction enzyme digestion verification
(No.1 are linearized T-vector, No.2 are C2-luxS fragment.)

Week6

The sequencing result showed there's a correct strain. So we can use the strain for the following experiment. We obtained the correct plasmid T-C2-luxS from DH5α. Then we got the fragment C2-luxS by digestion and gel extraction.

40μL digestion system
10X FastDigest Buffer 4μL
BamH Ⅰ 2μL
T-C2-luxS 25μL
ddH2O 9μL
Total 20μL
Reaction condition: 37℃ for 40 min

Ligated the C2-luxS to linearized plasmid pWH1520, and transformed it into DH5α.

20μL ligation system
10X DNA Ligase Buffer 2μL
T4 DNA Ligase 1μL
pMD19 T-Simple Vector 1μL
C2-luxS 5μL
ddH2O 11μL
Total 20μL
Reaction condition: 16℃ overnight

Extracted the plasmid pWH-C2-luxS from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.

Extracted the plasmid pWH-C2-luxS from JM110,and dealt with it by BamH Ⅰ methylase.

100μL methylation system
10X BamH Ⅰ methyltransferase Buffer 10μL
BamH Ⅰ methyltransferase 1μL
S-adenosylmethionine 0.5μL
pWH-C2-luxS 80μL
ddH2O 8.5μL
Total 100μL
Reaction condition: 37℃ for 1 hour

Transformed the plasmid into GR286 by electroporation.[Failed]

Selecting positive clones by colony PCR
(No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-C2-luxS into GR286)

Week7

This week, we tried to use different voltages to transform the plasmid. Sadly, all of these tries got bad results.

We considered whether the luxS gene is a little toxic for GR286, and the bacteria tends to refuse the gene when we added a strong promoter in front of it. So, we planned to use induced expression to reconstruction our expression vector.

The plasmid pWH1520 contains the strong xylA promoter originating, and transcription from this promoter is xylose inducible. So, the gene of interest carries its own ribosome binding sequence (RBS) and translation initiation codon. Based on these points, we redesigned primers.

Week8

We cloned luxS gene from GR286 using our new primers.

50μL PCR system X2
2X Taq Master Mix 25μL
YD-luxS-F 2μL
YD-luxS-R 2μL
Bacterium solution 2μL
ddH2O 19μL
Total 50μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
58℃ 30 sec 30 cycles
72℃ 40 sec
72℃ 10 min
16℃ ∞
Cloning luxS gene by PCR

Purified the luxS fragment by gel extraction, and ligated it into linearized pWH1520. Then, transformed the vector into DH5α.

40μL digestion system X2
10X FastDigest Buffer 4μL
BamH Ⅰ 2μL
pWH1520 25μL
ddH2O 9μL
Total 40μL
Reaction condition: 37℃ for 40 min
20μL ligation system
10X DNA Ligase Buffer 2μL
T4 DNA Ligase 1μL
linearized pWH1520 1μL
luxS 3μL
ddH2O 13μL
Total 20μL
Reaction condition: 16℃ overnight

Selected the positive clones by colony PCR.

20μL PCR system
2X Taq Master Mix 10μL
pWH-F 1μL
pWH-R 1μL
Bacterium solution 1μL
ddH2O 7μL
Total 20μL
PCR reaction condition
94℃ 10 min
94℃ 30 sec
58℃ 30 sec 30 cycles
72℃ 40 sec
72℃ 10 min
16℃ ∞

We chose 4 positive strains to culture overnight and extracted the plasmid. After restriction enzyme digestion verification, we sent them to sequencing.

20μL digestion system
10X FastDigest Buffer 2μL
BamH Ⅰ 1μL
pWH-luxs 10μL
ddH2O 7μL
Total 20μL
Reaction condition: 37℃ for 40 min
Restriction enzyme digestion verification
(No.1 are linearized pWH1520, No.2 are luxS fragments.)

Week9

The sequencing result showed there's three correct strains. So we can choose a correct strain for the following experiment. We extracted the correct plasmid pWH-luxS from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.

Extracted the plasmid pWH -luxS from JM110,and dealt with it by BamH Ⅰ methylase.

100μL methylation system
10X BamH Ⅰ methyltransferase Buffer 10μL
BamH Ⅰ methyltransferase 1μL
S-adenosylmethionine 0.5μL
pWH-C2-luxS 80μL
ddH2O 8.5μL
Total 100μL
Reaction condition: 37℃ for 1 hour

Transformed the plasmid into GR286 by electroporation, and selected positive clones.

Selecting positive clones by colony PCR

The construction of supplier was complished!

Week10

Our synthetic lsrACDB gene came back. We first used restriction-ligation method to ligate lsrACDB to plasmid pWH1520, but we failed to select positive after several tries.

20μL ligation system
10X DNA Ligase Buffer 2μL
T4 DNA Ligase 1μL
linearized pWH1520 1μL
lsrACDB 3μL
ddH2O 13μL
Total 20μL
Reaction condition: 16℃ overnight

Considering the lsrACDB gene is a large fragment (4500bp), we used ClonExpress technique cloning the gene again to improve the efficiency of ligation. We divided the lsrACDB sequence into two parts and cloned them separately. Then we ligated the two segments to the plasmid pWH1520 and transformed it into DH5α. After that, we used PCR to select the positive clones. However, we didn't get a good result.

Selecting positive clones by colony PCR
(No.6 is positive control, No.1-5 are experimental groups)

Week11

We learnt a new method called circular polymerase extension cloning (CPEC) for high-throughput cloning of complex and combinatorial DNA libraries, and we decided to use this method to try ligating our lsrACDB gene. It's encouraging that we succeeded to ligate the lsrACDB gene to the plasmid pHT-01.

Selecting positive clones by colony PCR
(No.3-6 are positive clones)
Restriction enzyme digestion verification
(No.2&3 are positive results.)

Week12

Since we have constructed "supplier" and part of "consumer", we decided to measure the growth curve to explore the function of our "controller".

Groups divided in this experiment
GR286 wild strain as control group
GR286ΔluxS GR286 without luxS gene
pWH-luxS luxS overexpression plasmid in GR286; without induced by xylose
pWH-luxS + xyl luxS overexpression plasmid in GR286; induced by xylose
pWH1520 empty plasmid in GR286 as control group
pHT-lsrACDB lsrACDB overexpression plasmid in GR286ΔluxS
pHT-01 empty plasmid in GR286ΔluxS as control group

Cultured media of our supplier was tested for the presence of AI-2 by inducing luminescence in Vibrio harveyi reporter strain BB170.

Week13

For our consumer, we should also clone the lsrK and lsrFG gene for phosphorylating and degrading AI-2. We used ClonExpress technique cloning the two genes and ligated them to plasmid pHT-01 successfully.

Restriction enzyme digestion verification
(No.1&3&4 are positive results. )

We cocultured the supplier with BB170 and tested the fluoresent intensity to explore the function of supplier. (negative result)

Week14

Week15

Week16

Week17

Week18

Week19

Week20