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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)
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
1. Restriction enzyme digestion -> purification -> ligation -> transformation
- P1,23L with E,X -> successful
- P1,7L with E,S -> successful
2. 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
3. transformation from iGEM Kit plate
- P1,7M -> failure
9/10
1. colony PCR -> preculture
- P1,7L + P1,23L
2. Miniprep
- P1,23L digested with E,X
- P1,7L digested with E,S
3. transformation
- P1,7M -> successful
9/11
1. preculture
- P1,7M
2. Restriction enzyme digestion
- P1,23L with E -> failure
- P1,7L with E -> failure
9/12
1. Miniprep
- P1,7L + P1,23L
9/13
1. Miniprep
- P1,7M
2. Restriction enzyme digestion
- P1,23L with X
- P1,7L with S
- pUC57 with NcoI
9/14
1. ligation
- P1,7L + P1,23L
9/15
1. Restriction enzyme digestion -> failure
- pUC57 with E
- P1,7L with E
- P1,7M with E
9/16
1. colony PCR
- P1,7L + P1,23L -> failure
9/17
1. Restriction enzyme digestion -> failure
- pUC57 with NcoI, XbaI
- P1,7L with E,S
2. PCR
- P1,7L(lacI)
- P1,14L(araC)
- P1,2B(GFP)
9/18
1. Restriction enzyme digestion
- pUC with NcoI, XbaI
- P1,7L with E,S
9/19
1. Ligation -> transformation
- P1,7L + P1,23L -> successful
2. colony PCR
- P1,7L + P1,23L -> failure
9/20
1. preculture
- P1,7M
9/21
1. PCR from E.coli genome
- lacI -> failure
2. PCR -> purification
- lacI(P1,7L)
- lacI(E.coli genome)
- araC
- GFP
3. transformation
- P1,7L + P1,23L
4. Miniprep
- P1,7M -> failure
9/22
1. PCR
- lacI(E.coli genome) -> successful
- GFP -> successful
2. sequencing
- lacI(P1,7L) -> failure
- lacI(E.coli genome) -> failure
3. homologous recombination -> transformation
- araC + pUC57
- lacI + pUC57
4. preculture
- P1,7L + P1,23L
9/23
1. sequencing
- lacI(P1,7L) -> failure
- lacI(E.coli genome) -> failure
2. Miniprep
- P1,7L + P1,23L
Since P1,7M is not available, we decided to use P3,21D instead (They both have p15A origin). However, as it turned out that P3,21D was also difficult to clone, we BBa_I7100(Courtesy of Chiba team).
9/24
1. sequencing
- lacI(P1,7L) -> successful
- lacI(E.coli genome) -> failure
- P1,7L + P1,23L -> failure
2. colony PCR -> preculture
- lacI
- araC
- GFP
3. Ligation -> transformation
- P3,21D + (P1,7L + P1,23L)
9/25
1. Miniprep
- araC + pUC57
- lacI + pUC57
- GFP +pUC57
9/26
1. colony PCR
- P1,7L + P1,23L -> successful
2. sequencing
- lacI -> successful
- araC -> failure
- GFP -> successful
- P1,7L + P1,23L
9/27
1. sequencing
- P1,7L + P1,23L -> successful
2. Miniprep
- I7100
9/28
1. sequencing
- lacI -> successful
- araC -> Sequencing was successful, but it turned out that different DNA has amplified, not araC.
- GFP -> successful
- araC + pUC57 -> failure
- lacI +pUC57 -> failure
- GFP + pUC57 -> failure
- P1,7L + P1,23L -> failure
2. Restriction enzyme digestion
- P3,21D with E,S
9/29
1. ligation -> preculture -> Miniprep
- P3,21D + (P1,7L + P1,23L)
2. sequencing -> failure
- araC + pUC57 -> failure
- lacI +pUC57 -> failure
- GFP + pUC57 -> failure
- P1,7L + P1,23L -> failure
9/30
1. Restriction enzyme digestion
- araC with E,S
- GFP with E,X
- lacI with E,S
October
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