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Team:Todai-Tokyo/Notebook/bioclock
From 2009.igem.org
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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)
Our Records
Although we are very interested in "bioclock", what to do in this iGEM project was really difficult to decide. After discussing and searching paper for few months, we finally decided to make "UV induced oscillator", as described on the Project page. The oscillator was based on the previous work, the switching device was added to control the oscillator as we want. As we were not quite sure whether this circuit really work, so we decided to analyze it in silico, by modeling.
Detailed experimental plan was settled near the end of July. Since the promoter of the oscillatory device was synthesized products and unavailable from any oforganism, we had it synthesized. It took almost a month until we receive the DNA!!
When making the oscillator, we needed the plasmid vector of p15A origin which has low copy numbers and ColE1 origin of high copy numbers in E.coli cells, as strict control of the total protein amount was required. Among iGEM parts, pSB3K3 plasmid vector had the p15A origin and BBa_J63010 had ColE1 origin.
ColE1 origin plasmid were successfully cloned from 2009 Spring Kit Plate1, 7D. Meanwhile, the cloning of p15A ori plasmid was somehow of extreme difficulty. The first parts we choose to clone was 2009 Spring Kit Plate1, 7M and start to clone it from August 6, but never had it been available. After numerous unfruitful trials, we gave up and decided to get it from 2009 Spring Kit Plate3, 21D on September 24, but this parts neither couldn't be cloned. Finally, Chiba team gave us the BBa_I7100 parts, and now we were able to use it. Come to think of it, it took so long to change our plan of what part to be cloned. It was perhaps due to the difficulty of sharing information. Since we are on four project at the same time, it was very hard for each member to fully catch up with all the experiments ongoing.
Other genes are also cloned after numerous failures. Although wiki freezes on 21 October, we are still trying to complete our experiments. You will see whether we can succeed or not at the Jamboree!!
July
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
- P1.14L(araC)
- P1.7L(lacI)
- P1.4E(cI)
- P1.3D(ColE1)
- P1.9C(p15A)
- P1.9G(p15A)
7/30
- P1.14L(araC)
- P1.7L(lacI)
- P3.21D
August
- 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
8/1
- pre-culture of plate1 4E and 9C
8/2
- Miniprep of plate1 4E and 9C
8/3
- Transformation of plata1-9C and 9G
8/5
- Transformation of plate1-9G
8/6
8/7
- Transformation of plate1-23L and plate 3-21D
8/8
- Transformation of plate1-23L and plate 3-21D
8/9
- pre-culture of plate1-23L and plate 3-21D
8/10
- Miniprep of Plate1- 23L and P3-21L
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
September
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
- 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
10/10
1. Infusion of cIII and pUC57
10/11
1. Restriction Enzyme digestion
- araC with E,S
- GFP with E,X
- lacI with E,S
2. colony PCR ->
10/12
1. preculture ->Miniprep
- cIII + pUC57 -> successful
10/13
1. PCR
- araC from E.coli genome
2. Restriction Enzyme digestion
- I7100 with E,S
10/14
1. E.coli araC(-) strain culture 2. PCR from E.coli genome
- araC
3. Ligation
- araC + pUC57
10/15
1. PCR from E.coli genome
- araC
10/16
1. PCR
- araC
- lacI
- GFP
- cIII + pUC57
2. Homologous recombination
- cI~cII + I7100
- N + I7100
10/17
change vector of araC, lacI, GFP from pUC57 to I7100
1. Restriction enzyme digestion
- araC + pUC57 with E,P
- GFP + pUC57 with E,P
- P1,22A(Its plasmid vector has the ColE1 ori) with E,P
2. Sequencing
- cIII
- araC + pUC57
3. Ligation
- lacI + I7100
- araC + P1,22A
- GFP + P1,22A
10/19
1. Restriction Enzyme digestion
- P1,22A with E,P
2. Ligation
- lacI + I7100
- araC + P1,22A
- GFP + P1,22A
2. minipreps
- cI~cII
3. colony PCR
- I7100+P-OL~N-double terminator
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