Team:Macquarie Australia/Notebook3ChlH
Notebook - ChlH Gene
This notebook includes the lab-work done to insert one specific gene into E. coli that is part of the Chlorophyll Biosynthesis Pathway.
The 4166 bp ChlH gene was engineered synthetically by Integrated DNA Technologies (IDT) into 3 gBlocks:
| G1-3 | P2 | G3-6 |
|---|---|---|
| 1678 bp | 980 bp | 1508 bp |
Thursday 6 August 2015
- Attempted assembly of ChlH fragments into CAM and KAN backbones via Gibson Assembly
- Amplified GA mix to check whether gblocks had successfully been assembled. 3 sets of primers were used (table 1).
- Ran amplicons on a 1% agarose gel with GelRed. If assembly was successful, a band around 4.2 kbp is expected.
- No DNA evident on gel
Table 1: Primers used for PCR
| Forward | Reverse |
|---|---|
| Vector forward (BBF) | Vector reverse (BBR) |
| ChlH forward | ChlH reverse |
| Vector forward 2 (BBVF2) | Vector reverse 2 (BBVR2) |
- Transformed 2 uL KAN and CAM assembly products into E. coli cells and plated 100 uL and 150 uL of transformants out on nutrient agar plates with appropriate KAN or CAM antibiotic
- Colonies present on all plates (table 2) but transformation efficiency was very low
- Lots of pink colonies - insert not present
Table 2: Colony counts of Gibson Assembly transformants
| Type | Colour | 100 uL plates | 150 uL plates | Total |
|---|---|---|---|---|
| CAM | Pink | 6 | 3 | 20 |
| White | 8 | 3 | ||
| KAN | Pink | 1 | 0 | 3 |
| White | 1 | 1 | ||
| Control | Pink | 0 | 5 | 12 |
| White | 0 | 7 |
Wednesday 12 August 2015
- Harvested white colonies and cultured them in LB broth with the appropriate antibiotics
- 1 white KAN colony
- 5 white CAM colonies
Thursday 13 August 2015
- Isolated plasmids from liquid cultures via GenElute Plasmid Miniprep Kit (Sigma-Aldrich). Quality and concentration of isolated DNA was assessed via NanoDrop and 1% agarose gel stained with GelRed (table 3).
Table 3: Concentration and purity of isolate plasmid DNA obtained from NanoDrop.
| Sample | Concentration (ng/uL) | 260/280 |
|---|---|---|
| CAM 1 | 155.3 | 1.95 |
| CAM 2 | 302.1 | 1.91 |
| CAM 3 | 253.5 | 1.94 |
| CAM 4 | 277.8 | 1.95 |
| CAM 5 | 119.5 | 1.85 |
| KAN 1 | 87.2 | 1.91 |
- Performed double restriction digests with EcoRI and EcoRI + PstI, and ran product on a gel to determine if ChlH had been successfully assembled by last weeks Gibson Assembly.
Thursday 27 August 2015
- Nanodrop results:
- 260/280 values indicate isolated
- Performed double restriction digests with EcoRI and EcoRI + PstI, and ran product on a gel to determine if ChlH had been successfully assembled by last weeks Gibson Assembly.
- Results of gel indicated single bands for the double digest, suggesting that the restriction digest was unsuccessful. The EcoRI restriction site may have disappeared.