Team:NTU-Singapore/Notebook

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 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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