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The 4166 bp ChlH gene was engineered synthetically by Integrated DNA Technologies (IDT) into 3 gblocks:
| 1(G13) |
1678 bp |
| 2 (P2) |
980 bp |
| 3 (3-6) |
1508 bp |
Thursday 6th 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.
Table 1: Primers used for PCR
| 1. |
Vector forward (BBF) |
Vector reverse (BBR) |
| 2. |
ChlH forward |
ChlH reverse |
| 3. |
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
| |
100 uL plates |
150 uL plates |
Total |
|
pink |
white |
pink |
white |
|
| CAM |
6 |
8 |
3 |
3 |
20 |
| KAN |
1 |
1 |
0 |
1 |
3 |
| Control |
0 |
0 |
5 |
7 |
12 |
Wednesday 12th August 2015
- Harvested white colonies and cultured them in LB broth with the appropriate antibiotics
- 1 white KAN colony
- 5 white CAM colonies
Thursday 13th August 2015
- Isolated plasmids from liquid cultures via GenElute Plasmid Miniprep Kit (Sigma-Aldrich) for PCR to check for successful cloning of ChlH. 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.
|
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 |
- 260/280 values indicate isolated DNA was of fairly high quality.
- Performed first round of 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.
- Ran restriction products on a 1% gel stained with GelRed. Gel indicated that assembly was unsuccessful.
- Assembly may have failed due to poor overlap between part G13 and the KAN vector (11 bp) and the KAN biobrick vector. Potentially there is also insufficient overlap between the KAN vector and part 3-6 (19 bp). Optimal overlap for Gibson Assembly is 15 - 30 bp.
Wednesday 19th August 2015
- Attempted new assembly of ChlH via restriction digest and ligation for parts G13, 3-6 and KAN vector
- 3-6-------------------------KAN vector-----------------------G13
- Expect an approximately 5kb band on gel if assembly is successful
- Method:
- 2x Master Digest Mix
| Master Digest Mix |
4 uL 10x Buffer for EcoRI and PstI
14 uL water
1 uL EcoRI
1 uL PstI |
- Digest protocol
- 2.5 uL linearised vector
- 1 uL each gblock (G13 and 3-6)
- 4.5 uL digest master mix
- Incubate at 37 C for 1 hour and heat inactivate at 80 C for 15 min
- Ligation Protocol
- 9 uL 2x T4 Ligase buffer to digest mix
- 1 uL T4 DNA ligase
- incubate at 25 C for 20 min, 37 C for 20 min and heat inactivate at 80 C for 10 min
- Ran digest products on a 1% agarose gel stained with GelRed
- Approx 5kb band evident - sees as though gblocks G13 and 3-6 were ligated to the linearized KAN vector.
- Attempted assembly of P2 with other gblocks and KAN vector via Gibson Assembly
-
-
and gibson assembly for part P2.
Thursday 27th August 2015
- 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.
Wednesday 3rd September 2015
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