Team:Todai-Tokyo/Notebook/bioclock

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*cut plate1-23L(double terminator) and insert plate1-7L(lacI) in it<BR>
*cut plate1-23L(double terminator) and insert plate1-7L(lacI) in it<BR>
*PCR of cI~cII<BR>
*PCR of cI~cII<BR>
 +
 +
'''8/27'''
 +
1. Amplify following parts with PCR (Pfu Ultra) → electroporesis → column purification (*1) <BR>
 +
#P1, 7L (lacI, 1140bp) <BR>
 +
Primer 5’ : F_E_7M_lacI+tag5’, <BR>
 +
Primer 3’ : F_S_7M_tag+lacI3’ <BR>
 +
 +
#P1, 4E (cI, 740bp) <BR>
 +
Primer 5’ : F_E_7M_c2+c15’, <BR>
 +
Primer 3’ : F_S_7M_c2+c13’ <BR>
 +
 +
2. Cut Plate 1, 23L (double terminator) with EcoRI to make lacI + double teminator <BR>
 +
 +
'''8/28'''
 +
1. Sequencing of (*1) parts of 8/27 -> Successful, but not perfect. Need to be re-sequenced <BR>
 +
2. Amplify following parts with PCR <BR>
 +
#P1,7L <BR>
 +
Primer 5’ : F_Xh_lacI+tag5’ <BR>
 +
Primer 3’ : F_Xh_lacI+tag3’ <BR>
 +
 +
3. Sequencing P1,23L (double teminator) -> failure <BR>
 +
 +
'''8/30'''
 +
1. re-sequencing of (*1) parts -> failure <BR>
 +
 +
'''9/1'''
 +
1. re-sequencing of (*1) parts -> failure <BR>
 +
 +
'''9/3'''
 +
1. Cut following parts with EcoRI, SpeI Restriction enzymes to make RBS + lacI +doubleterminator <BR>
 +
#P1, 23L <BR>
 +
#P1,7L -> It was a mistake, P1,7L should have been cut with EcoRI and XbaI <BR>
 +
 +
'''9/4'''
 +
1. Restriction enzyme reaction <BR>
 +
#P1,23L with EcoRI and XbaI <BR>
 +
 +
2. Ligation of P1,7L and P1,23L -> failure, must be tried again <BR>
 +
 +
'''9/5'''
 +
Amplify following genes from genome of bacteriophage lambda with PCR <BR>
 +
#cI~cII <BR>
 +
#OL~N <BR>
 +
 +
'''9/6'''
 +
1. Restriction enzyme reaction -> ligation (*2) <BR>
 +
P1,7L with E, S <BR>
 +
P1,23L with E, X <BR>
 +
 +
'''9/7'''
 +
1. Transformation of following DNA DH5alpha E.coli cells -> failure <BR>
 +
# (*2) ligation product -> failure, must be tried again <BR>
 +
# P1,7M -> failure, P1,7M might be denatured, since we forgot to put it in refrigerator, and left it at room temperature <BR>
 +
# P1, 4E -> successful <BR>
 +
# pUC57 -> successful <BR>
 +
 +
*since P1,7M was denatured, all experiments below turned to be failure <BR>
 +
 +
2. Restriction enzyme reaction <BR>
 +
# P1, 7M with E, S (*3) -> failure <BR>
 +
 +
3. Transform following genes (amplified on 9/5) into (*3) P1,7M vector with homologous recombination <BR>
 +
# cI~cII -> failure <BR>
 +
# OL~N -> failure <BR>
 +
 +
'''9/8'''
 +
1.
=='''October'''==
=='''October'''==

Revision as of 07:05, 20 October 2009

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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-

Methods:
1. Clone the following genes from Bacillus subtilis into a biobrick vector.


  1. plate1,13B
  2. plate1,13L
  3. 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

  1. P1.14L(araC)
  2. P1.7L(lacI)
  3. P1.4E(cI)
  4. P1.3D(ColE1)
  5. P1.9C(p15A)
  6. P1.9G(p15A)

7/30

Miniprep

  1. P1.14L(araC)
  2. P1.7L(lacI)
  3. 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)

  1. P1, 7L (lacI, 1140bp)

Primer 5’ : F_E_7M_lacI+tag5’,
Primer 3’ : F_S_7M_tag+lacI3’

  1. P1, 4E (cI, 740bp)

Primer 5’ : F_E_7M_c2+c15’,
Primer 3’ : F_S_7M_c2+c13’

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

  1. 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

  1. P1, 23L
  2. P1,7L -> It was a mistake, P1,7L should have been cut with EcoRI and XbaI

9/4 1. Restriction enzyme reaction

  1. 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

  1. cI~cII
  2. 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

  1. (*2) ligation product -> failure, must be tried again
  2. P1,7M -> failure, P1,7M might be denatured, since we forgot to put it in refrigerator, and left it at room temperature
  3. P1, 4E -> successful
  4. pUC57 -> successful
  • since P1,7M was denatured, all experiments below turned to be failure

2. Restriction enzyme reaction

  1. P1, 7M with E, S (*3) -> failure

3. Transform following genes (amplified on 9/5) into (*3) P1,7M vector with homologous recombination

  1. cI~cII -> failure
  2. OL~N -> failure

9/8 1.

October

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