From 2009.igem.org

(Difference between revisions)

Revision as of 08:21, 20 October 2009

Home	The Team	The Project	Parts Submitted to the Registry	Modeling	Notebook	Protocols	Ethics

the notebook

Aim: Create E.coli that show oscillatory gene expression pattern (colE1 ori vector)-placI/araC-araC+ssra tag-double terminator-placI/araC-GFP+ssra tag-double(sample)

Plan

Aim: Create E.coli that show oscillatory gene expression pattern (colE1 ori vector)-placI/araC-araC+ssra tag-double terminator-placI/araC-GFP+ssra tag-double terminator- (p15A ori)-placI/araC-lacI+ssra tag-double terminator-

Methods:
1. Clone the following genes from Bacillus subtilis into a biobrick vector.

plate1,13B
plate1,13L
plate2,1H

2. Make a gene network that express oscillatory pattern, using these genes.

7/7

Cloning the parts
preculture of the Biobrick parts for Miniprep

7/8

Cloning the parts
Miniprep of E.coli cells containing Biobrick parts with Promega, Wizard Plus SV Miniprep DNA Purification System

failure

7/9

Cloning the parts
Miniprep again of E.coli cells containing Biobrick parts with Promega, Wizard Plus SV Miniprep DNA Purification System

7/29

Miniprep

P1.14L(araC)
P1.7L(lacI)
P1.4E(cI)
P1.3D(ColE1)
P1.9C(p15A)
P1.9G(p15A)

7/30

Miniprep

P1.14L(araC)
P1.7L(lacI)
P3.21D

August/September

create plasmid including EcoRI restriction site, XbaI restriction site, placI/araC, XhoI restriction site, NcoI restriction site, double terminator, SpeI restriction site and PstI restriction site.→pUC57

-E-X-placI/araC-XhoI-NcoI-double terminator-S-P-

cut pUC57 by XhoI and NcoI

→insert GFP, araC or lacI
(GFP:2006 plate1-16E,araC and lacI:2009 iGEM distribution)

cut plate1-23L(double terminator) and insert plate1-7L(lacI) in it
PCR of cI~cII

8/27

1. Amplify following parts with PCR (Pfu Ultra) → electroporesis → column purification (*1)

P1, 7L (lacI, 1140bp)

Primer 5’ : F_E_7M_lacI+tag5’,
Primer 3’ : F_S_7M_tag+lacI3’

P1, 4E (cI, 740bp)

Primer 5’ : F_E_7M_c2+c15’,
Primer 3’ : F_S_7M_c2+c13’

2. Cut Plate 1, 23L (double terminator) with EcoRI to make lacI + double teminator

8/28 1. Sequencing of (*1) parts of 8/27 -> Successful, but not perfect. Need to be re-sequenced
2. Amplify following parts with PCR

P1,7L

Primer 5’ : F_Xh_lacI+tag5’
Primer 3’ : F_Xh_lacI+tag3’

3. Sequencing P1,23L (double teminator) -> failure

8/30 1. re-sequencing of (*1) parts -> failure

9/1 1. re-sequencing of (*1) parts -> failure

9/3 1. Cut following parts with EcoRI, SpeI Restriction enzymes to make RBS + lacI +doubleterminator

P1, 23L
P1,7L -> It was a mistake, P1,7L should have been cut with EcoRI and XbaI

9/4 1. Restriction enzyme reaction

P1,23L with EcoRI and XbaI

2. Ligation of P1,7L and P1,23L -> failure, must be tried again

9/5 Amplify following genes from genome of bacteriophage lambda with PCR

cI~cII
OL~N

9/6 1. Restriction enzyme reaction -> ligation (*2)
P1,7L with E, S
P1,23L with E, X

9/7 1. Transformation of following DNA DH5alpha E.coli cells -> failure

(*2) ligation product -> failure, must be tried again
P1,7M -> failure, P1,7M might be denatured, since we forgot to put it in refrigerator, and left it at room temperature
P1, 4E -> successful
pUC57 -> successful

since P1,7M was denatured, all experiments below turned to be failure

2. Restriction enzyme reaction

P1, 7M with E, S (*3) -> failure

3. Transform following genes (amplified on 9/5) into (*3) P1,7M vector with homologous recombination

cI~cII -> failure
OL~N -> failure

9/8 1.

October

Home	The Team	The Project	Parts Submitted to the Registry	Modeling	Notebook	Protocols	Ethics

@@ Line 80: / Line 80: @@
 *PCR of cI~cII<BR>
-'''8/27'''
+=='''8/27'''==
 .	Amplify following parts with PCR (Pfu Ultra) → electroporesis → column purification (*1) <BR>
 #P1, 7L (lacI, 1140bp) <BR>

Team:Todai-Tokyo/Notebook/bioclock