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 T7promoterpSB1K3
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
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