Team:NTU-Singapore/Notebook

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* Antibiotic stock preparation
* Antibiotic stock preparation
* Inoculation
* Inoculation
 +
 +
 +
<u>'''20th  June'''</u>
 +
* Our first Plasmid Extraction (PE)!
 +
 +
 +
<u>'''22nd June'''</u>
 +
* First Double Digestion (DD) and Gel Check!
 +
 +
 +
<u>'''23rd June '''</u>
 +
* PE & DD of the registry constructs….
 +
 +
 +
<u>'''24th June '''</u>
 +
*Our direct synthesized parts finally arrived. We now have CHE (cholesterol esterase), IFP(Infrared protein 1.4) and pNO (NO sensing promoter).
 +
 +
 +
<u>'''26th June'''</u>
 +
*PE , DD and Gel check for the direct synthesized gene sequence.
 +
 +
 +
<u>'''27th June  '''</u>
 +
*Gel Check for CHE, IFP and pNO
 +
 +
 +
==July==
 +
 +
 +
<u>'''1st July'''</u>
 +
* PE for plaqIQ, J23101, J23119, B0034(rbs), B0015(Term-T for short), B0034-GFP-B0015
 +
 +
 +
<u>'''2nd July'''</u>
 +
* PE for pNO, IFP, CHE
 +
* DD of pNO, J23119, J23101, rbs-GFP-T
 +
* Our first ligation!
 +
 +
 +
<u>'''3rd July'''</u>
 +
* DD of CHE, IFP and B0015, plaqIQ and plux.
 +
* Ligation.
 +
 +
 +
<u>'''4th July'''</u>
 +
* PE: pNO-GFP; J23101-GFP; J23119-GFP; HO1
 +
* DD: HO1
 +
 +
 +
<u>'''7th July'''</u>
 +
* PE: CHE, IFP, placIQ
 +
* DD:CHE-T, IFP-T. HO1, Term
 +
* Ligation
 +
 +
 +
<u>'''8th July'''</u>
 +
* DD: pNO-GFP; J23101-GFP; J23119-GFP
 +
 +
 +
<u>'''9th July'''</u>
 +
* PE
 +
* DD: ptet. r-CHE-T, r-HO1, r-IFP-T
 +
* Ligation: r-HO1 +r-IFP-T
 +
 +
 +
<u>'''10th July'''</u>
 +
* PE
 +
* DD: ptet; r-CHE-T, Term, tetR
 +
 +
 +
<u>'''11th July'''</u>
 +
* PE
 +
* DD: r-CHE-T; r-HO1-r-IFP-T;CHE-T; r-CHE-T
 +
 +
 +
<u>'''13th July'''</u>
 +
* DD: ptet; r-CHE-T; r-HO1-r-IFP-T; TetR; Term
 +
* Ligation: ptet + r-CHE-T; ptet + r-HO1-r-IFP-T; TetR + Term
 +
 +
 +
<u>'''15th July'''</u>
 +
* PE
 +
* DD: pTet; rbs; IFP; CHE; TetR-T
 +
 +
 +
<u>'''16th July'''</u>
 +
* DD: pTet; r-CHE-T; Inv; r-HO1-r-IFP-T; Term; TetR
 +
* Ligation
 +
 +
 +
<u>'''18th July'''</u>
 +
* PE
 +
* DD and Single Digestion (SD) :r-HO1-r-IFP-T (because they have the same size)
 +
 +
 +
<u>'''20th July'''</u>
 +
* DD:pTet; J23119; r-HO1-r-IFP-T; TetR; Term; placIQ; inv
 +
* Ligation: pTet +r-HO1-r-IFP-T; J23119 + r-HO1-r-IFP-T; placIQ +Inv; TetR +Term
 +
* PE 
 +
 +
 +
<u>'''23rd July'''</u>
 +
* PE
 +
* DD:J23119; Inv; r-HO1; r-GFP-T; pTet; placIQ-Inv; TetR-T
 +
 +
 +
<u>'''24th July'''</u>
 +
* PE
 +
* DD: J23119; r-CHE-T; pTet; inv;r-HO1-r-IFP-T; ptet-IFP
 +
* Ligation: J23119+r-CHE-T; ptet+r-CHE-T; ptet+r-HO1-r-IFP-T; J23119+Inv
 +
 +
 +
<u>'''28th July'''</u>
 +
* PE
 +
* DD: r-HO1; r-GFP-T; J23119;r-CHE-T;r-HO1-r-IFP-t; ptet; r-CHE-T
 +
* Ligation: r-HO1 +r-GFP-T; J23119 +r-CHE-T; J23119 + r-HO1-r-IFP-T; pTet +r-CHE-T; pTet +r-GFP-T
 +
 +
 +
<u>'''30th July'''</u>
 +
* PE
 +
*DD : J23119-r-CHE-T; ptet-r-CHE-T; J23119-r-HO1-r-IFP-T;  ptet-r-HO1-r-IFP-T; r-HO1-r-GFP-T; J23119; pTet; TetR; rbs
 +
* Ligation: r-HO1 +r-GFP-T; J23119 +r-CHE-T; J23119+r-HO1-IFP-T; ptet+r-CHE-T; ptet+r-GFP-T   
 +
 +
 +
== August ==
 +
 +
 +
<u>'''4th August'''</u>
 +
* DD :rbs; NorR
 +
 +
 +
<u>'''5th August'''</u>
 +
* PE
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''12th August'''</u>
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''14th August'''</u>
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''17th August'''</u>
 +
* DD
 +
* Our first SDS-PAGE—J23119-r-CHE-T; pTet-r-CHE-T
 +
 +
 +
<u>'''18th August'''</u>
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''21st August'''</u>
 +
* DD
 +
* Characterization of J23119
 +
 +
 +
<u>'''24th August'''</u>
 +
* PCR verification –HO1, pTet constructs
 +
 +
 +
<u>'''25th August'''</u>
 +
* pTet characterization
 +
 +
 +
<u>'''26th August'''</u>
 +
* DD
 +
 +
 +
<u>'''27th August'''</u>
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''28th August'''</u>
 +
* PCR verification – CHE constructs
 +
* DD
 +
* Ligation
 +
 
 +
 +
<u>'''29th August'''</u>
 +
* DD
 +
* Ligation
 +
* PCR verification –IFP constructs
 +
 +
 +
<u>'''31st August'''</u>
 +
* PCR verification –HO1, CHE constructs
 +
* DD
 +
* Ligation 
 +
 +
 +
==September==
 +
 +
 +
<u>'''1st September'''</u>
 +
* PCR verification –IFP, NorR, pNO
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''2nd September'''</u>
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''3rd September'''</u>
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''4th September'''</u>
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''6th September'''</u>
 +
* PCR --J23119-r-NorR-T
 +
 +
 +
 +
<u>'''7th September'''</u>
 +
* DD,
 +
* Ligation
 +
 +
 +
 +
<u>'''8th September'''</u>
 +
* PCR verification –IFP, HO1 constructs
 +
 +
 +
 +
<u>'''9th September'''</u>
 +
* PCR verification –HO1 constructs
 +
 +
 +
 +
<u>'''10th September'''</u>
 +
* DD
 +
* Ligation
 +
 +
 +
 +
<u>'''11th September'''</u>
 +
* PCR verification –NorR, IFP, HO1 constructs
 +
* Ligation
 +
 +
<u>'''12thS eptember'''</u>
 +
* PCR verification – NorR, IFP, HO1 constructs
 +
 +
 +
<u>'''14thSeptember'''</u>
 +
* PCR verification – IFP, HO1 constructs
 +
 +
 +
<u>'''15th September'''</u>
 +
* DD
 +
* Ligation
 +
* SDS-Page
 +
 +
 +
<u>'''16th September'''</u>
 +
* pNO characterization
 +
 +
 +
<u>'''17th September'''</u>
 +
* Ligation
 +
 +
 +
<u>'''18th September'''</u>
 +
* DD
 +
* Ligation
 +
 +
 +
<u>'''19th September'''</u>
 +
* DD
 +
 +
 +
<u>'''20th September'''</u>
 +
* Ligation
 +
 +
 +
<u>'''22nd September'''</u>
 +
* PCR verification –IFP, HO1
 +
* pNO characterization
 +
 +
 +
<u>'''23rd September'''</u>
 +
* PCR verification --CHE
 +
 +
 +
<u>'''24th September'''</u>
 +
* PCR verification –CHE, HO1
 +
* DD
 +
* Ligation
 +
* Optimization of CHE characterization experiment
 +
 +
 +
<u>'''26th September'''</u>
 +
* DD
 +
* Ligation
 +

Latest revision as of 19:39, 21 October 2009

Experiment Timeline


June

18th June

  • Preparation of LB medium
  • Amplification of parts from registry (pTet, TetR, HO1, Low copy plasmid)


19th June

  • Antibiotic stock preparation
  • Inoculation


20th June

  • Our first Plasmid Extraction (PE)!


22nd June

  • First Double Digestion (DD) and Gel Check!


23rd June

  • PE & DD of the registry constructs….


24th June

  • Our direct synthesized parts finally arrived. We now have CHE (cholesterol esterase), IFP(Infrared protein 1.4) and pNO (NO sensing promoter).


26th June

  • PE , DD and Gel check for the direct synthesized gene sequence.


27th June

  • Gel Check for CHE, IFP and pNO


July

1st July

  • PE for plaqIQ, J23101, J23119, B0034(rbs), B0015(Term-T for short), B0034-GFP-B0015


2nd July

  • PE for pNO, IFP, CHE
  • DD of pNO, J23119, J23101, rbs-GFP-T
  • Our first ligation!


3rd July

  • DD of CHE, IFP and B0015, plaqIQ and plux.
  • Ligation.


4th July

  • PE: pNO-GFP; J23101-GFP; J23119-GFP; HO1
  • DD: HO1


7th July

  • PE: CHE, IFP, placIQ
  • DD:CHE-T, IFP-T. HO1, Term
  • Ligation


8th July

  • DD: pNO-GFP; J23101-GFP; J23119-GFP


9th July

  • PE
  • DD: ptet. r-CHE-T, r-HO1, r-IFP-T
  • Ligation: r-HO1 +r-IFP-T


10th July

  • PE
  • DD: ptet; r-CHE-T, Term, tetR


11th July

  • PE
  • DD: r-CHE-T; r-HO1-r-IFP-T;CHE-T; r-CHE-T


13th July

  • DD: ptet; r-CHE-T; r-HO1-r-IFP-T; TetR; Term
  • Ligation: ptet + r-CHE-T; ptet + r-HO1-r-IFP-T; TetR + Term


15th July

  • PE
  • DD: pTet; rbs; IFP; CHE; TetR-T


16th July

  • DD: pTet; r-CHE-T; Inv; r-HO1-r-IFP-T; Term; TetR
  • Ligation


18th July

  • PE
  • DD and Single Digestion (SD) :r-HO1-r-IFP-T (because they have the same size)


20th July

  • DD:pTet; J23119; r-HO1-r-IFP-T; TetR; Term; placIQ; inv
  • Ligation: pTet +r-HO1-r-IFP-T; J23119 + r-HO1-r-IFP-T; placIQ +Inv; TetR +Term
  • PE


23rd July

  • PE
  • DD:J23119; Inv; r-HO1; r-GFP-T; pTet; placIQ-Inv; TetR-T


24th July

  • PE
  • DD: J23119; r-CHE-T; pTet; inv;r-HO1-r-IFP-T; ptet-IFP
  • Ligation: J23119+r-CHE-T; ptet+r-CHE-T; ptet+r-HO1-r-IFP-T; J23119+Inv


28th July

  • PE
  • DD: r-HO1; r-GFP-T; J23119;r-CHE-T;r-HO1-r-IFP-t; ptet; r-CHE-T
  • Ligation: r-HO1 +r-GFP-T; J23119 +r-CHE-T; J23119 + r-HO1-r-IFP-T; pTet +r-CHE-T; pTet +r-GFP-T


30th July

  • PE
  • DD : J23119-r-CHE-T; ptet-r-CHE-T; J23119-r-HO1-r-IFP-T; ptet-r-HO1-r-IFP-T; r-HO1-r-GFP-T; J23119; pTet; TetR; rbs
  • Ligation: r-HO1 +r-GFP-T; J23119 +r-CHE-T; J23119+r-HO1-IFP-T; ptet+r-CHE-T; ptet+r-GFP-T


August

4th August

  • DD :rbs; NorR


5th August

  • PE
  • DD
  • Ligation


12th August

  • DD
  • Ligation


14th August

  • DD
  • Ligation


17th August

  • DD
  • Our first SDS-PAGE—J23119-r-CHE-T; pTet-r-CHE-T


18th August

  • DD
  • Ligation


21st August

  • DD
  • Characterization of J23119


24th August

  • PCR verification –HO1, pTet constructs


25th August

  • pTet characterization


26th August

  • DD


27th August

  • DD
  • Ligation


28th August

  • PCR verification – CHE constructs
  • DD
  • Ligation


29th August

  • DD
  • Ligation
  • PCR verification –IFP constructs


31st August

  • PCR verification –HO1, CHE constructs
  • DD
  • Ligation


September

1st September

  • PCR verification –IFP, NorR, pNO
  • DD
  • Ligation


2nd September

  • DD
  • Ligation


3rd September

  • DD
  • Ligation


4th September

  • DD
  • Ligation


6th September

  • PCR --J23119-r-NorR-T


7th September

  • DD,
  • Ligation


8th September

  • PCR verification –IFP, HO1 constructs


9th September

  • PCR verification –HO1 constructs


10th September

  • DD
  • Ligation


11th September

  • PCR verification –NorR, IFP, HO1 constructs
  • Ligation

12thS eptember

  • PCR verification – NorR, IFP, HO1 constructs


14thSeptember

  • PCR verification – IFP, HO1 constructs


15th September

  • DD
  • Ligation
  • SDS-Page


16th September

  • pNO characterization


17th September

  • Ligation


18th September

  • DD
  • Ligation


19th September

  • DD


20th September

  • Ligation


22nd September

  • PCR verification –IFP, HO1
  • pNO characterization


23rd September

  • PCR verification --CHE


24th September

  • PCR verification –CHE, HO1
  • DD
  • Ligation
  • Optimization of CHE characterization experiment


26th September

  • DD
  • Ligation




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