Team:Peking S/lab/notebook/wdq

Dingqiao Wen's Notebook

Summary

I'm responsible for part of the molecular cloning of the chemical wires and characterizing the properties of quorum sensing repressors.

Contents

• June, 2011

• July, 2011

• August, 2011

• September, 2011

• October, 2011

June

[TOP]

6.22 - 6.25

Design primers.

6.26 - 6.27

Culture streptomycin griseus strain, which is the source of afsA, arpA, padpA.

6.28

afsA, arpA PCR using different annealing temperature, with gradient 4 ℃.

Identify them by 1% agarose gel electrophoresis.

Excise the gel slice and extract the fragments.

Second PCR using the products of gel extraction.

Electrophoresis PCR reaction system in 1.5% agarose gel.

Double digest the products of gel extraction by EcoR1-HF and Xba1.

Double digest the plasmid of B0015 by EcoR1-HF and Spe1.

Ligase afsA and arpA to the vector of B0015.

6.29

Transform the ligation reaction system.

Sythesis the genes of CAI-1 system using DNAWorks assembly and PadpA PCR.

Identify them by 1% agarose gel electrophoresis.

Excise the gel slice of pqrr, J23106+B0034+luxU, PadpA and extract the fragments.

ligase the products of gel extraction to the vector pEASY-Blunt.

6.30

Transform the ligation reaction system.

Pick six clones each plate and shave at 37℃ to amplify the bacteria.

July

[TOP]

7.1

Miniprep of the plasmids which contain pqrr, J23106+B0034+luxU, padpA gene respectively and send for sequencing.

7.3

Get the result of sequencing and the sequence of J23106+B0034+luxU has a mutation which need to be corrected. Design primers for J23106+B0034+luxU mutation.

7.5

Get mutation primers and mutate the plasmid using TaKaRa MutanBEST Kit.

7.6

Pick six clones each plate and shave at 37℃ to amplify the bacteria. Miniprep of the plasmids.

7.7

Send the plasmid of J23106+B0034+luxU mutants for sequencing.

7.8 - 7.15

As for the initial results of DNAWorks assembly for cqsS, cqsA, luxO were not good, we changed the reaction conditions and tried many other alternative method. However, the consequence still remain disappointing. So we decide to get the strain of Vibrio cholerae and use the method of PCR:

cqsS, cqsA, luxO PCR using different annealing temperature, with gradient 4 ℃.

Identify them by 1% agarose gel electrophoresis.

Excise the gel slice of cqsS, cqsA, luxO and extract the fragments.

ligase the products of gel extraction to the vector pEASY-Blunt.

Transformation.

7.16

Send the plasmid of cqsS, cqsA, luxO for sequencing.

Since there are some EcoR1, Pst1, Xba1 restriction sites in the original sequence of cqsS, cqsA, luxO, We design primers for point mutatiion to eliminate the restriction sites.

7.18

Get mutation primers and mutate the plasmid using TaKaRa MutanBEST Kit.

7.19

Pick five clones each plate and shave at 37℃ to amplify the bacteria.

Miniprep of the plasmids.

7.20

Send the plasmid of cqsS, cqsA, luxO mutants for sequencing.

7.22

Get the result of sequencing.

Make the second point mutation in cqsS.

7.23

Pick five clones in cqsS mutants plate and shave at 37℃ to amplify the bacteria.

Miniprep of the plasmids.

7.24

Send the plasmid of cqsS mutants for sequencing.

August

[TOP]

7.25 - 8.12

1.Put the double digested fragments of J23106+B0034+luxU, cqsS, luxO in the ligation reaction system in 16℃ for 2-4 hours, then add the EcoR1 and Pst1 double-digested vector pSB1C3 for 4 hours.

Transform the ligation reaction system.

16-18 hours later, pick six to ten clones in the plate and shave at 37℃ to amplify the bacteria. Miniprep of the plasmids.

2. Construct pBAD+B0034+cqsA+B0015 plasmid in pSB1AC3 backbone.

3. Construct Pqrr+B0030+GFP+ssrA degredation tag in pSB4A5 backbone.

8.13 - 8.20

Characterize CAI-1 system

8.21 - 8.26

1.Construct J23106+B0034+lasR+B0015 plasmid and J23106+B0034+rhlR+B0015 plasmid in pSB1A3 backbone.

2. design and order the primers for the quorum sensing repressor system

8.27 - 8.31

pcr the artificial promoters onto the gfp+ssrA fragment, Xba1 and Pst1 digested, and ligase with pSB4K5 backbone.

September

[TOP]

9.1

pcr the artificial promoters onto the gfp+ssrA fragment, Xba1 and Pst1 digested, and ligase with pSB4K5 backbone.

9.6 - 9.17

1.characterize the property of artificial promoters, rhlA and las_CS using 3oc4-HSL and 3oc12-HSL respectively, and rhlA promoter works well while the las_CS promoter seems have no evident function.

2. construct J23106+B0034+luxR+B0015+ lux-inverter-promoter+gfp+ssrA degredation tag plasmid and characterize its property using 3oc6-HSL.

9.18 - 9.26

transform the artificial promoters (including rhlA, las-inverter promoter, rhl-inverter promoter, and lasB_OP2 promoter) +gfp ssrA to pSB1C3 backbone.

9.27 - 09.30

construct J23106+B0034+lasR/rhlR+B0015+inverter-promoters+gfp+ssrA degredation tag plasmid and characterize their property using 3oc4-HSL and 3oc6-HSL respectively.

October

[TOP]

10.1 - 10.4

construct J23106+B0034+lasR/rhlR+B0015+inverter-promoters+gfp+ssrA degredation tag plasmid and characterize their property using C4-HSL and 3OC6-HSL respectively.

10.7

10.8

10.9

10.10

10.11

10.12

10.18 - 10.21

10.21 - 10.25