Team:Todai-Tokyo/Notebook/bioclock

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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)

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

  1. Restriction enzyme digestion -> purification -> ligation -> transformation
  • P1,23L with E,X -> successful
  • P1,7L with E,S -> successful
  1. 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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