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℃