Team:Peking S/lab/notebook/lhc

My major task is molecule cloning of generators and corresponding receivers for chemical wires such as A-factor. In addition, characterization of some logic gates like AND GATE as well as constructing a three input gate using new signaling molecules is my major contribution in the later stage of our poject. What is more, I am also responsible for the direction and manufacture of the CG animation about invertors.

July

[TOP]

7.1

1. Learn the basic experiment skills including transformation and ligation and digestion..

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(2011).

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”.

7.14

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 coding for"Pbad+GFP+Psal+GFP", which can operate as an demonstration of 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 neither of two.

7.16

1. Success ligation of the supD+terminator in the downstream of arac+pbad+supD+nahr+psal.

2. Cut the backbones including psb3T5,psb4K5.

7.17

1. Positively transform the plasmid from part 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 jointed”.

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 psb3T5.

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.

7.26 - 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 ribosome binding sites, so we start to PCR the T7ptag again after figuring out what happen.

August

[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 mutated.

September

[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.

October

[TOP]

Mon	Tue	Wed	Thu	Fri	Sat	Sun
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25	-	-	-	-	-	-

@@ Line 4: / Line 4: @@
-<font size=6> <font color="#FFFFFF">Handuo Shi's Notebook</font></FONT>
+<font size=6> <font color="#FFFFFF">Hanchi Lin's Notebook</font></FONT>
-== '''summary''' ==
+== '''Summary''' ==
-blahblah...
+My major task is molecule cloning of generators and corresponding receivers for chemical wires such as A-factor. In addition, characterization of some logic gates like AND GATE as well as constructing a three input gate using new signaling molecules is my major contribution in the later stage of our poject. What is more, I am also responsible for the direction and manufacture of the CG animation about invertors.
 =='''Contents'''==
@@ Line 81: / Line 81: @@
 |}
 [<html><a href="#top">TOP</a></html>]
-===7.3 - 7.11===
+===7.1===
-Synthesis of pqrr, cqsS, luxU, luxO & cqsA by DNAWorks.
-===7.12===
+.  Learn the basic experiment skills including transformation and ligation and digestion..
-PCR cqsS, luxU, luxO & cqsA from newly-obtained genomes.
+===7.2===
-===7.13===
+.  Electrophoresis enzyme digestion reaction system is a 1.5% agarose gel.
-cqsA & luxU obtained from genomes, sent for sequencing.
+.  Excise the gel slice and extract the DNA fragments.
-pqrr obtained from DNAWorks.
+.  Ligation of insert DNA fragments for 3 hours.
+.  Transform the products of ligation.
+===7.3===
+.  DNA double digestion of the plasmid including pbad.
+.  Positively transform the part 1-14N(2011).
+.  Ligation of pbad and gfp.
+===7.4===
+.  Transform the products of ligation of pbad and gfp and cultivate the plate overnight.
+===7.5===
+.  pick five clones cultivated overnight on the plate.
+.  Using the clones to PCR to verify the DNA fragments are connected correctly.
+.  And cultivate the bacteria in LB for 10 hours.
+.  According the result of PCR, choose the correct cloning to miniprep the plasminds.
+===7.6===
+.  Get the ligation of gfp and nahr+psal+gfp transformed.
+.  Pick up the clones and cultivate the bacteria overnight.
+===7.7 - 7.10===
+.  Get the plasmid coding for “Pbad+GFP+Psal+GFP”.
+.  DNA double digestion of part 1-5G(Xbal and Pstl) and part 1-7C(Xbal and Pstl).
+.  DNA double digestion of plasmid coding for “Pt7+GFP” and plasmid coding for “T7ptag”.
 ===7.14===
-Mutate luxU.
+.  Ligation of T7ptag and terminator.
-PCR for cqsA.
+.  Transform the ligation product.
-cqsS obtained from genomes.
+.  After pick some clones to PCR, cultivate the same clones.
+.  Successfully get the plasmid coding for"Pbad+GFP+Psal+GFP", which can operate as an demonstration of OR GATE.
 ===7.15===
-cqsS obtained for sequencing.
+.  Find the accurately ligation to miniprep.
+.  Try to link the supD+terminator before pt7+GFP+terminator, fail.
+.  Spread the bacteria with plasmid pbad+gfp+psal+gfp on four different plates with salicylic acid, arabinose, both of two and neither of two.
 ===7.16===
-Mutation of luxO.
+.  Success ligation of the supD+terminator in the downstream of arac+pbad+supD+nahr+psal.
-luxU mutant obtained.
+.  Cut the backbones including psb3T5,psb4K5.
+===7.17===
+.  Positively transform the plasmid from part 1-15P.
+.  Try to link the rbs+arac+pbad+supD+nahr+psal+supD with pt7+gfp+terminator, fail.
 ===7.18===
-===7.19===
+.  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===
+.  We decide to use a method called ”three pieces jointed”.
+.  Get the plasmid rbs+arac+pbad+supD+nahr+psal+supD double digested (Ecorl Spel) and plasmid pt7+gfp+terminator double digested(Xbal Pstl).
-===7.20===
+.  Connect the two DNA fragments with psb3T5.
+.  Get the newly gained plasmid coding for pqrr by PCR and get it double digested.
 ===7.21===
-===7.22===
+.  Miniprep the pqrr+T7ptag+terminator and double digest it to switch another backbone.
+===7.26 - 7.27===
-===7.23===
+.  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.
-===7.29===
+.  Pick the clones and we deliver the plasmid for sequencing once more.
-===7.30===
+===7.28 - 7.30===
-===7.31===
+.  Find that pqrr+T7ptag+terminator has two ribosome binding sites, so we start to PCR the T7ptag again after figuring out what happen.
-hello
 ==August==
@@ Line 186: / Line 244: @@
 ===8.1===
-===8.2===
+.  Since the sequencing result has come, we are sure that we has got our plasmids validly assembled.
-===8.4===
+.  Plasmids including:
+pqrr+T7ptag+terminator (rbs: b0034);.
+pqrr+T7ptag+terminator (rbs: b0035);
-===8.5===
+===8.2 - 8.3===
-===8.6===
+.  Send the rbs+arac+pbad+supD+nahr+psal+supD+pt7+gfp+terminator plasmid for sequencing.
-===8.7===
+.  Connect the pqrr+cqsa.
-===8.8===
+===8.4===
-===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===
+.  Ultimately, get all the obligable plasmid clones assembled correctly.
-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===
+.  Switch the plasmid to corresponding backbone.
-Ligation of pc+1-8O.   (pBAD+supD)+(plux'+T7ptag (6))+4K5 sequencing wrong!  Digest (pBAD+supD).
-===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===
+.  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).
-===8.31===
+.  The primary result are not satisfying, hence, start to have the rbs strength mutated.
-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==
@@ Line 312: / Line 322: @@
 |}
 [<html><a href="#top">TOP</a></html>]
-===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===
-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===
+．  Use other promoters to substitute the original promoter for construct other AND GATE.
-===9.28===
+===9.6 - 9.30===
-===9.29===
+Use PlacI invertor to construct a NOT GATE.
-===9.30===
+Not mainly in charge of some specific mission, help others to promot the progress of our project.
 ==October==

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Team:Peking S/lab/notebook/lhc

From 2011.igem.org

Latest revision as of 00:17, 6 October 2011

Summary

Contents

July

7.1

7.2

7.3

7.4

7.5

7.6

7.7 - 7.10

7.14

7.15

7.16

7.17

7.18

7.19 - 7.20

7.21

7.26 - 7.27

7.28 - 7.30

August

8.1

8.2 - 8.3

8.4

8.5 - 8.20

September

9.1 - 9.5

9.6 - 9.30

October

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

Mon	Tue	Wed	Thu	Fri	Sat	Sun
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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
-	-	-	-	-	-	-

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	-	-	-	-

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	-	--

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4	5	6	7	8	9	10
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25	-	-	-	-	-	-