Team:PKU Beijing/Notebook/AND Gate 1/Core/Min Lin

Notebook > AND Gate 1 > Core > Min Lin's Note

2009.8.1
Transformation: J23100-tetR-tetP and LuxR-LuxP-SupD

PCR assessment for the J23100-tetR- tetP and LuxR-LuxP-SupD Overnight

2009.8.2
PCR product->GEL

Number2-6 J23100-tetR-tetP is correct. Number1 of LuxR-LuxP-SupD is correct.

Digestion of the J23100-tetR-tetP With EcoRI and SpeI overnight

2009.8.3
01:30 Help Zhangguosheng pick colonies and PCR

10:00 GEL





Enzyme Digest of the J23100-tetR-tetP with EcoRI and SpeI

GEL Purification. Ligation: J23100-tetR-tetP ES insert E0840 EX vector and SupD-terminator EX vector

18:30 Transformation.

2009.8.4
PCR assessment

Shake the BL21 strain transformed with T7promoter-GFP by WangHao Induce with IPTG. The induced one has fluorescence

2009.8.5
Pick standard part I0500 Phusion PCR

GEL purification and then cut with EcoRI and SpeI. Purify from digestion.

2009.8.6
Ligation of I0500(ES) to SupD-terminator and E0840 vector.

Transformation

In order to get a Kan resistant back bone, I transformed the pSB1K3 part from plate1-7A.

2009.8.7
Shake the 1-7A in the incubator. PCR assessment of the I0500-GFP clone. But it is no need to care about which is right, cause Gaorencheng has worked out a AraC and PBad promoter that works well.

2009.8.8
Miniprep the 1-7A plasmid. Digest with EcoRI and PstI

GEL purification. Store the backbone.

Ligation T7promoter-GFP EP insert pSB1K3 EP backbone

Transformation

2009.8.9
Pick colonies to do PCR assessment of the T7promoterpSB1K3 The 3 colonies are correct ones.

Induction of the LuxR-LuxP-GFP A gradient of concentration: 5uM, 2uM, 100nM, 10nM, 1nM, 0 Use flocytometry to detect the fluorescence. It shows a ten fold induction. However the basal is very high.

Miniprep the LuxR-LuxP-SupD plasmid and the T7promoter- GFP-pSB1K3 plasmid.

2009.8.11
10:00 Help Zhangguosheng with his GEL

15:00 Enzyme Digestion of the LuxR-LuxP-T7ptag by XbaI and PstI

2009.8.13
Miniprep the Low copy plasmids, and digest with EcoRI and PstI.

Digest the LuxR-LuxP-SupD plasmid with SpeI and PstI, CIAP and then purify.

Ligation: Insert: XP digestion of AraC-T7ptag insert (9x rbs) Vector: LuxR-LuxP-SupD(SP)

2009.8.14
Shake the 3 counter plasmid in the incubator. Miniprep the SupD-terminator plasmid. Digest with EcoRI and XbaI to make vectors.

Help Wushuke induce the pLac-RFP-JM109. Miniprep the 3 counter plasmid.

2009.8.15
11:00 Phusion PCR of the 3 counter plasmid. GEL purification. Digest with EcoRI and PstI

Ligation: pSB1K3 backbone 1 ul T3 pol insert 7ul