Team:Peking S/lab/notebook/shd

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Handuo Shi's Notebook


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Contents

June

Mon Tue Wed Thu Fri Sat Sun
- - 1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 29 - - -

[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.

July

Mon Tue Wed Thu Fri Sat Sun
- - - - 1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
- - - - - - -

[TOP]

7.3 - 7.11

Synthesis of pqrr, cqsS, luxU, luxO & cqsA by DNAWorks.

7.12

PCR cqsS, luxU, luxO & cqsA from newly-obtained genomes.

7.13

cqsA & luxU obtained from genomes, sent for sequencing.

pqrr obtained from DNAWorks.

7.14

Mutate luxU.

PCR for cqsA.

cqsS obtained from genomes.

7.15

cqsS obtained for sequencing.

7.16

Mutation of luxO.

luxU mutant obtained.

7.18

7.19

7.20

7.21

7.22

7.23

7.29

7.30

7.31

hello

August

Mon Tue Wed Thu Fri Sat Sun
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31 - - - -

[TOP]

8.1

8.2

8.4

8.5

8.6

8.7

8.8

8.9

8.10

8.11

8.12

8.13

8.14

8.15

8.16

8.17

8.18

Transform of luxR(1-8O, 936bp, I0462), plux'(1-14P, 30bp, R0061), luxI(3-14A, 711bp, K081015)

8.19

Retransform of plux', luxI; pc+luxR

8.20

Ligation of plux'+T7ptag(2673 bp)+terminator, pT7 (I719005) +luxI.

8.21

CAI-1 induce of the CAI-1 system.

8.22

Ligation of pc+luxR, transform of luxI(1-14C, 661bp, C0261)

8.23

pT7+luxI 1,3,5 sequencing right. Digestion of luxI(1-14C)

8.24

Ligation of pT7+luxI(no terminator), (pBAD+supD)+(plux'+T7ptag (6))+4K5.

8.25

pc+luxR(1-8O) was wrong in part! Transform of luxR(2-4O,799bp,J37033)

8.26

CAI-1 induce of the CAI-1 system.

8.27

pT7+luxI(no term). No. 1, 3 sequencing right. Ligation of pT7+luxI + GFP(ssrA tag) for sequencing. Colony PCR, (1) 1,3,4,5 (3)1,2,3,4. PCR for 1-4O, 1-8O(in 2009 Distribution). Digest 1-2M(RBS). Transform of pc(J23100, 1-18C), luxR(1-4O).

8.28

Ligation of pc+1-8O. (pBAD+supD)+(plux'+T7ptag (6))+4K5 sequencing wrong! Digest (pBAD+supD).

8.29

pT7+luxI+GFP(ssrA tag) sequencing wrong (No luxI?!) Ligation again for 3A (psb1C3). Ligation for RBS+luxR(1-4O). Ligation (pBAD+supD)+(plux'+T7ptag (6))+4K5 again. (btw T7ptag sequencing right).

8.30

(pBAD+supD)+(plux'+T7ptag (6))+4K5 16, 32 for sequencing. pT7+luxI+GFP(ssrA tag) colony PCR: (1). 7, 8, 10; (3). 1, 3, 4, 5, 6, 7, 9, 10. Digest RBS+luxR(1-4O).

8.31

pT7+luxI+GFP(ssrA tag) Digest check: (1). 7, 8, 10; (3). 4, 6, 7, 9. for sequencing. Ligation of pC+RBS+luxR send for sequencing.

September

Mon Tue Wed Thu Fri Sat Sun
- - 1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 - --

[TOP]

9.1

RBS+luxR sequencing right in the end! Religation of (pBAD+supD)+(plux'+T7ptag (6))+4K5 and (pBAD+supD)+(plux'+T7ptag (6))+3C5, doubt whether (pBAD+supD) has been right, redigest.

9.2

pT7+luxI+GFP(ssrA tag) sequencing right. Ligation of (pT7+luxI+GFP)+(pC+RBS+luxR) psB1A3. Religation of (pBAD+supD)+(plux'+T7ptag (6))+4K5 and (pBAD+supD)+(plux'+T7ptag (6))+3C5. (pBAD+supD)+(plux'+T7ptag (6))+4K5 (43) sequencing right!

9.3

(pT7+luxI+GFP)+(pC+RBS+luxR) psB1A3 colony PCR right. Double transform for induce.

9.4

Induce of the feedback circuit.

9.5

Induce of the feedback circuit. (pT7+luxI+GFP)+(pC+RBS+luxR) psB1A3 sent for sequencing.

9.6

9.7

9.8

9.9

9.12

9.13

9.14

9.15

9.16

9.17

9.21

9.22

9.23

9.24

9.25

9.26

9.27

9.28

9.29

9.30

October

Mon Tue Wed Thu Fri Sat Sun
- - - - 1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 - - - - - -

[TOP]

10.1

10.3

10.4

10.5

10.7

10.8

10.9

10.10

10.11

10.12

10.13

10.15

10.16-10.21

10.21-10.25

 

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