- Preparation of LB medium
- Amplification of parts from registry (pTet, TetR, HO1, Low copy plasmid)
- Antibiotic stock preparation
- Our first Plasmid Extraction (PE)!
- First Double Digestion (DD) and Gel Check!
- PE & DD of the registry constructs….
- Our direct synthesized parts finally arrived. We now have CHE (cholesterol esterase), IFP(Infrared protein 1.4) and pNO (NO sensing promoter).
- PE , DD and Gel check for the direct synthesized gene sequence.
- Gel Check for CHE, IFP and pNO
- PE for plaqIQ, J23101, J23119, B0034(rbs), B0015(Term-T for short), B0034-GFP-B0015
- PE for pNO, IFP, CHE
- DD of pNO, J23119, J23101, rbs-GFP-T
- Our first ligation!
- DD of CHE, IFP and B0015, plaqIQ and plux.
- PE: pNO-GFP; J23101-GFP; J23119-GFP; HO1
- DD: HO1
- PE: CHE, IFP, placIQ
- DD:CHE-T, IFP-T. HO1, Term
- DD: pNO-GFP; J23101-GFP; J23119-GFP
- DD: ptet. r-CHE-T, r-HO1, r-IFP-T
- Ligation: r-HO1 +r-IFP-T
- DD: ptet; r-CHE-T, Term, tetR
- DD: r-CHE-T; r-HO1-r-IFP-T;CHE-T; r-CHE-T
- 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
- DD: pTet; rbs; IFP; CHE; TetR-T
- DD: pTet; r-CHE-T; Inv; r-HO1-r-IFP-T; Term; TetR
- DD and Single Digestion (SD) :r-HO1-r-IFP-T (because they have the same size)
- 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
- DD:J23119; Inv; r-HO1; r-GFP-T; pTet; placIQ-Inv; TetR-T
- 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
- 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
- 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
- DD :rbs; NorR
- Our first SDS-PAGE—J23119-r-CHE-T; pTet-r-CHE-T
- Characterization of J23119
- PCR verification –HO1, pTet constructs
- pTet characterization
- PCR verification – CHE constructs
- PCR verification –IFP constructs
- PCR verification –HO1, CHE constructs
- PCR verification –IFP, NorR, pNO
- PCR --J23119-r-NorR-T
- PCR verification –IFP, HO1 constructs
- PCR verification –HO1 constructs
- PCR verification –NorR, IFP, HO1 constructs
- PCR verification – NorR, IFP, HO1 constructs
- PCR verification – IFP, HO1 constructs
- pNO characterization
- PCR verification –IFP, HO1
- pNO characterization
- PCR verification --CHE
- PCR verification –CHE, HO1
- Optimization of CHE characterization experiment