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Team:Todai-Tokyo/Notebook/bioclock
From 2009.igem.org
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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)
Contents |
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-
Also sub-clone gene that constructs the UV induced switch into p15A ori plasmid
-lacI-cI-OR(operator region of cI and cro)-cro-Nut(N utilization; N binding sequence)cII-cIII-OL(operator region of N)-N(enhancer of cII)
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)
- P1, 4E (cI, 740bp)
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
- Restriction enzyme digestion -> purification -> ligation -> transformation
- P1,23L with E,X -> successful
- P1,7L with E,S -> successful
- preculture
- pUC57 vector
- P1,4E -> Afterward, this part was turned out to be unusable. We sub-cloned cI~cII gene from the genome of bacteriophage lambda
9/10
October
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