Team:Peking S/lab/notebook/lhc

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===7.3 - 7.11===
+
===7.1===
-
Synthesis of pqrr, cqsS, luxU, luxO & cqsA by DNAWorks.
+
-
===7.12===
+
1.  the PCR primers is delivered and get the product: arac+Pbad+supD+nahr+Psal by PCR.
 +
2.  double digestion of the PCR product.
-
PCR cqsS, luxU, luxO & cqsA from newly-obtained genomes.
+
===7.2===
-
===7.13===
+
1.  Electrophoresis enzyme digestion reaction system is a 1.5% agarose gel.
 +
2.  Excise the gel slice and extract the DNA fragments.
 +
3.  Ligation of insert DNA fragments for 3 hours.
 +
4.  Transform the products of ligation.
-
cqsA & luxU obtained from genomes, sent for sequencing.
+
===7.3===
-
pqrr obtained from DNAWorks.
+
1.  DNA double digestion of the plasmid including Pbad.
 +
2.  Positively transform the part 1-14N.
 +
3.  Ligation of Pbad and GFP.
-
===7.14===
+
===7.4===
-
Mutate luxU.
+
1.  Transform the products of ligation of Pbad and GFP and cultivate the plate overnight.
-
PCR for cqsA.
+
===7.5===
 +
 
 +
1.  pick five clones cultivated overnight on the plate.
 +
2.  Using the clones to PCR to verify the DNA fragments are connected correctly.
 +
3.  And cultivate the bacteria in LB for 10 hours.
 +
4.  According the result of PCR, choose the correct cloning to miniprep the plasminds.
 +
 
 +
===7.6===
 +
 
 +
1.  Get the ligation of GFP and nahr+Psal+GFP transformed.
 +
2.  Pick up the clones and cultivate the bacteria overnight.
 +
 
 +
===7.7 - 7.10===
 +
 
 +
1.  Get the plasmid coding for “Pbad+GFP+Psal+GFP”.
 +
2.  DNA double digestion of part 1-5G(Xbal and Pstl) and part 1-7C(Xbal and Pstl).
 +
3.  DNA double digestion of plasmid coding for “Pt7+GFP” and plasmid coding for “T7ptag”.
 +
 
 +
===7.11===
 +
 
 +
===7.12===
 +
 
 +
===7.13===
 +
 
 +
===7.14===
-
cqsS obtained from genomes.
+
1.  Ligation of T7ptag and terminator.
 +
2.  Transform the ligation product.
 +
3.  After pick some clones to pcr, cultivate the same clones.
 +
4.  Successfully get the plasmid Pbad+GFP+Psal+GFP, which can severs as an or gate.
===7.15===
===7.15===
-
cqsS obtained for sequencing.
+
1.  Find the accurately ligation to miniprep.
 +
2.  Try to link the supD+terminator before Pt7+GFP+terminator, fail.
 +
3.  Spread the bacteria with plasmid Pbad+GFP+Psal+GFP on four different plates with salicylic acid, arabinose, both of two and none of two.
===7.16===
===7.16===
-
Mutation of luxO.
+
1.  Success ligation of the supD+terminator in the stern of arac+Pbad+supD+nahr+Psal.
 +
2.  Cut the backbones including 3T5,4K5.
-
luxU mutant obtained.
+
===7.17===
 +
 
 +
1.  Positively transform the plasmid from parts 1-15P.
 +
2.  Try to link the rbs+arac+Pbad+supD+nahr+Psal+supD with Pt7+GFP+terminator, fail.
===7.18===
===7.18===
-
===7.19===
+
1.  Find out that the both rbs+arac+Pbad+supD+nahr+Psal+supD and Pt7+GFP+terminator have a long sequence,thus it is difficult to connect them in a usual way.
-
===7.20===
+
===7.19 - 7.20===
 +
 
 +
1.  We decide to use a method called ”three pieces connected”.
 +
2.  Get the plasmid rbs+arac+Pbad+supD+nahr+Psal+supD double digested (Ecorl Spel) and plasmid Pt7+GFP+terminator double digested(Xbal Pstl).
 +
3.  Connect the two DNA fragments with 3T5.
 +
4.  Get the newly gained plasmid coding for Pqrr by PCR and get it double digested.
===7.21===
===7.21===
 +
 +
1.  Miniprep the Pqrr+T7ptag+terminator and double digest it to switch another backbone.
===7.22===
===7.22===
Line 124: Line 169:
===7.23===
===7.23===
-
===7.29===
+
===7.24===
-
===7.30===
+
===7.25===
-
===7.31===
+
===7.26 - 7.27===
-
hello
+
 +
1.  Find the concentration of rbs+arac+Pbad+supD+nahr+Psal+supD+Pt7+GFP+terminator which has been sent for sequencing is too low to sequencing.
 +
2.  Pick the clones and we deliver the plasmid for sequencing once more.
 +
 +
===7.28 - 7.30===
 +
 +
1.  Find that Pqrr+T7ptag+terminator has two rbs, so we start to PCR the T7ptag again.
 +
 +
===7.31===
==August==
==August==
Line 186: Line 238:
===8.1===
===8.1===
-
===8.2===
+
1. Since the sequencing result has come, we are sure that we has got our plasmids validly assembled.
 +
2.  Plasmids including:
 +
Pqrr+T7ptag+terminator (rbs: b0034);.
 +
Pqrr+T7ptag+terminator (rbs: b0035);
-
===8.4===
+
===8.2 - 8.3===
-
===8.5===
+
1.  Send the rbs+arac+Pbad+supD+nahr+Psal+supD+Pt7+GFP+terminator plasmid for sequencing.
 +
2.  Connect the Pqrr+Cqsa.
-
===8.6===
+
===8.4===
-
 
+
-
===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===
+
1.  Ultimately, get all the obligable plasmid clones assembled correctly.
-
Ligation of pc+1-8O.   (pBAD+supD)+(plux'+T7ptag (6))+4K5 sequencing wrong! Digest (pBAD+supD).
+
2.  Switch the plasmid to corresponding backbone.  
-
===8.29===
+
===8.5 - 8.20===
-
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===
+
1.  Assist in the induction of XOR gate.
-
(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).
+
2.  The primary result are not satisfying, hence, start to have the rbs strength mutation.
-
===8.31===
+
===8.21 - 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==
==September==
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-
===9.1===
+
===9.1 - 9.5===
-
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===
+
1．  Use other promoters to substitute the original promoter for construct other AND GATE.
-
Induce of the feedback circuit.
+
-
===9.5===
+
===9.6 - 9.30===
-
Induce of the feedback circuit.  (pT7+luxI+GFP)+(pC+RBS+luxR) psB1A3 sent for sequencing.
+
-
===9.6===
+
Use PlacI invertor to construct a NOT GATE.
 +
Not mainly in charge of some specific mission, help others to promot the progress of our project.
===9.7===
===9.7===

Revision as of 13:09, 1 October 2011


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Handuo Shi's Notebook


summary

blahblah...

Contents

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]

7.1

1. the PCR primers is delivered and get the product: arac+Pbad+supD+nahr+Psal by PCR. 2. double digestion of the PCR product.

7.2

1. Electrophoresis enzyme digestion reaction system is a 1.5% agarose gel. 2. Excise the gel slice and extract the DNA fragments. 3. Ligation of insert DNA fragments for 3 hours. 4. Transform the products of ligation.

7.3

1. DNA double digestion of the plasmid including Pbad. 2. Positively transform the part 1-14N. 3. Ligation of Pbad and GFP.

7.4

1. Transform the products of ligation of Pbad and GFP and cultivate the plate overnight.

7.5

1. pick five clones cultivated overnight on the plate. 2. Using the clones to PCR to verify the DNA fragments are connected correctly. 3. And cultivate the bacteria in LB for 10 hours. 4. According the result of PCR, choose the correct cloning to miniprep the plasminds.

7.6

1. Get the ligation of GFP and nahr+Psal+GFP transformed. 2. Pick up the clones and cultivate the bacteria overnight.

7.7 - 7.10

1. Get the plasmid coding for “Pbad+GFP+Psal+GFP”. 2. DNA double digestion of part 1-5G(Xbal and Pstl) and part 1-7C(Xbal and Pstl). 3. DNA double digestion of plasmid coding for “Pt7+GFP” and plasmid coding for “T7ptag”.

7.11

7.12

7.13

7.14

1. Ligation of T7ptag and terminator. 2. Transform the ligation product. 3. After pick some clones to pcr, cultivate the same clones. 4. Successfully get the plasmid Pbad+GFP+Psal+GFP, which can severs as an or gate.

7.15

1. Find the accurately ligation to miniprep. 2. Try to link the supD+terminator before Pt7+GFP+terminator, fail. 3. Spread the bacteria with plasmid Pbad+GFP+Psal+GFP on four different plates with salicylic acid, arabinose, both of two and none of two.

7.16

1. Success ligation of the supD+terminator in the stern of arac+Pbad+supD+nahr+Psal. 2. Cut the backbones including 3T5,4K5.

7.17

1. Positively transform the plasmid from parts 1-15P. 2. Try to link the rbs+arac+Pbad+supD+nahr+Psal+supD with Pt7+GFP+terminator, fail.

7.18

1. Find out that the both rbs+arac+Pbad+supD+nahr+Psal+supD and Pt7+GFP+terminator have a long sequence,thus it is difficult to connect them in a usual way.

7.19 - 7.20

1. We decide to use a method called ”three pieces connected”. 2. Get the plasmid rbs+arac+Pbad+supD+nahr+Psal+supD double digested (Ecorl Spel) and plasmid Pt7+GFP+terminator double digested(Xbal Pstl). 3. Connect the two DNA fragments with 3T5. 4. Get the newly gained plasmid coding for Pqrr by PCR and get it double digested.

7.21

1. Miniprep the Pqrr+T7ptag+terminator and double digest it to switch another backbone.

7.22

7.23

7.24

7.25

7.26 - 7.27

1. Find the concentration of rbs+arac+Pbad+supD+nahr+Psal+supD+Pt7+GFP+terminator which has been sent for sequencing is too low to sequencing. 2. Pick the clones and we deliver the plasmid for sequencing once more.

7.28 - 7.30

1. Find that Pqrr+T7ptag+terminator has two rbs, so we start to PCR the T7ptag again.

7.31

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

1. Since the sequencing result has come, we are sure that we has got our plasmids validly assembled. 2. Plasmids including: Pqrr+T7ptag+terminator (rbs: b0034);. Pqrr+T7ptag+terminator (rbs: b0035);

8.2 - 8.3

1. Send the rbs+arac+Pbad+supD+nahr+Psal+supD+Pt7+GFP+terminator plasmid for sequencing. 2. Connect the Pqrr+Cqsa.

8.4

1. Ultimately, get all the obligable plasmid clones assembled correctly. 2. Switch the plasmid to corresponding backbone.

8.5 - 8.20

1. Assist in the induction of XOR gate. 2. The primary result are not satisfying, hence, start to have the rbs strength mutation.

8.21 - 8.31

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 - 9.5

1． Use other promoters to substitute the original promoter for construct other AND GATE.

9.6 - 9.30

Use PlacI invertor to construct a NOT GATE. Not mainly in charge of some specific mission, help others to promot the progress of our project.

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

 

Retrieved from "http://2011.igem.org/Team:Peking_S/lab/notebook/lhc"