"
Team:Cambridge/Experiments/Assembly of Reflectin Constructs
From 2011.igem.org
This is a placeholder. We should fill it in.
Contents |
Construct Design
Primer Design
We should mention expected lengths of products here.
Assembly: first attempt
PCR
In the first round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3 and GA4 constructs.
- We performed PCR using Phusion Hot Start DNA Polymerase in 20 μl reaction volume.
- The time profile used in the PCR machine was the following:
| Hold | 95°C | 2 min | |
| Cycling | Denaturing | 95°C | 10 s |
| Annealing | 55°C | 20 s | |
| Elongation | 72°C | 150 s |
We decided to use the 55°C annealing temperaure, although the predicted temperature for most primers is 5-10°C higher, because of a low annealing temperature of the VF2 primer.
- Primers and template DNA provided by our supervisor Paul served as a positive control, but eventually we did not detect any products on the gels.
The pictures below present result of gel electrophoresis of PCR products.
- In most cases position of a band matches the expected length of DNA fragment. The only exception are GA1-2 and GA3-2 products. According to the position on the gel the length of these DNA fragments is 4-5kb, whereas the predicted length is 3.5kb. Our hypothesis is that we were provided [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] backbone instead of [http://partsregistry.org/Part:pSB1A3 pSB1A3]backbone.
- For GA1-1, GA2-1, GA3-1 and GA4-1 we obtained two bands: 1000bp and 400bp, with the latter resulting from non-specific priming most probably. We extracted the two bands for GA1-1, GA2-1 and GA4-1 products, labelling the 1000kb and 400bp fragments GAX-1a and GAX1-b respectively.
- The molecular weight marker that we used in all gels is HyperLadder I, which produces regularly spaced bands ranging from 200 to 10,000bp.
For the gel extraction of DNA we followed the protocol, assuming that one slice of gel is 100μl.
Gibson Assembly
We conducted Gibson Assembly of GA1, GA2, GA3 and GA4 constructs according to [Team:Cambridge/Protocols/Gibson_Assembly#Practice | the protocol]. The volumes of Master Mix and solutions of amplified DNA were the following:
| GA1 | GA2 | GA3 | GA4 |
|---|---|---|---|
| 15µl Master Mix | 15µl Master Mix | 15µl Master Mix | 15µl Master Mix |
| 2.5µl GA1-1a | 1.67µl GA2-1a | 2.5µl GA3-1 | 1.67µl GA4-1a |
| 2.5µl GA1-2 | 1.67µl GA2-2 | 2.5µl GA3-2 | 1.67µl GA4-2 |
| 1.67µl GA2-3 | 1.67µl GA4-3 |
Transformation
We transformed competent E.coli cells according to [Team:Cambridge/Protocols/Transformation_of_E.Coli | the following protocol]. We cultured each class of transformants on four different plates.
| scope="col" width="200" | GA1 and GA3 | GA2 and GA4 |
|---|---|---|
| Plate 1 (10μl of cell suspension) | LB + ampicillin | LB + kanamycin |
| Plate 2 (100μl of cell suspension) | LB + ampicillin | LB + kanamycin |
| Plate 3 (10μl of cell suspension) | LB + ampicillin + arabinose | LB + kanamycin + arabinose |
| Plate 4 (100μl of cell suspension) | LB + ampicillin + arabinose | LB + kanamycin + arabinose |
Results
Diagnostics
Assembly: second attempt
PCR
In the second round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3, GA4 as well as GA5 and GA6 constructs.
We ran PCR products on a 1% agarose gel to separate amplified products from template DNA and primers, as well as to check how efficient and specific the amplification process was.
- All components of GA5 and GA6 constructs produced clear fairly thick bands with positions matching expected lengths. No non-specific bands on GA5-1 (Reflectin A2) and GA6-1 (Reflectin 1B) lanes were observed.
- The molecular weight marker that we used in all gels is HyperLadder I, which produces regularly spaced bands ranging from 200 to 10,000bp.
Gibson Assembly
Transformation
Results
What next?
