Team:Peking S/lab/notebook/qx

Xiao Qin's Notebook

summary

In our project, my job is mainly to construct the competitor module, as well as characterize its performance. I also have a partner, Chen Yiwei, to fulfill this task together.

Contents

• July, 2011

• August, 2011

• September, 2011

• October, 2011

July

7.1 - 7.7

We did four rounds of PCR to get the ptsG1+gfp. The gfp here refers to BBa_E0840 from the iGEM 2011 Parts Kit. In each step, we added around 30bp of the ptsG1’s 5’ untranslated region (5’ UTR) to the former product, and we finally got the construct for the fused protein. We sequenced the plasmids and it turned out to be correct.

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