Team:NTU-Singapore/Notebook

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