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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)

1 Plan
- 1.1 7/7
- 1.2 7/8
- 1.3 7/9
- 1.4 7/29
- 1.5 7/30
- 1.6 August/September
- 1.7 8/27
- 1.8 8/28
- 1.9 8/30
- 1.10 9/1
- 1.11 9/3
- 1.12 9/4
- 1.13 9/5
- 1.14 9/6
- 1.15 9/7
- 1.16 9/8
- 1.17 9/10
- 1.18 9/11
- 1.19 9/12
- 1.20 9/13
- 1.21 9/14
- 1.22 9/15
- 1.23 9/16
- 1.24 9/17
- 1.25 9/18
- 1.26 9/19
- 1.27 9/20
- 1.28 9/21
- 1.29 9/22
- 1.30 9/23
- 1.31 October

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