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Team:Cambridge/Experiments/Assembly of Reflectin Constructs
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Contents |
Construct Design
Primer Design
We should mention expected lengths of products here - initial PCR reactions as well as colony PCR.
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 results of the 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 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 the following protocol. We cultured each class of transformants on four different plates.
| GA1 and GA3 | GA2 and GA4 | ||
| Plate 1 non-induced | (10μl of cell suspension) | LB + ampicillin | LB + kanamycin |
| Plate 2 non-induced | (100μl of cell suspension) | LB + ampicillin | LB + kanamycin |
| Plate 3 induced | (10μl of cell suspension) | LB + ampicillin + arabinose | LB + kanamycin + arabinose |
| Plate 4 induced | (100μl of cell suspension) | LB + ampicillin + arabinose | LB + kanamycin + arabinose |
After overnight incubation at 37°C of transformed E.coli cells, we could see colonies on only some of our plates. We examined successful plates under a fluorescence microscope to check if the cells had been transformed with a carry-through of the template DNA used in the initial PCR reactions. The risk of contaminating Gibson Assembly reactions with template DNA was fairly high because circular DNA of around 4kb was likely to co-localize with the linear PCR products.
| Type of plasmid | Expected phenotype of transformed colonies | |
| LB + Antibiotic | LB + Antibiotic + Arabinose | |
| Reflectin A1 expression plasmid | No fluorescence | No fluorescence |
| I13520:pSB1A3 plasmid | No fluorescence | RFP red fluorescence |
| J69511:pSB3K3 plasmid | GFP green fluorescence | GFP green fluorescence |
These are the results of the examination of transformants:
| GA1 | one culture on LB + ampicillin 10μl plate | no fluorescence detected |
| GA2 | no colonies observable | - |
| GA3 | colonies on all plates | no fluorescence detected |
| GA4 | colonies on all plates | green fluorescence on induced and non-induced plates |
Colony PCR
We decided to conduct colony PCR of E.coli transformed with GA1 and GA3 constructs in order to confirm successful assembly of the plasmids.
- We prepared overnight liquid cultures of GA3 and GA1 transformants, both in LB + amplicillin and LB + amplicillin + arabinose medium. We picked 6 colonies, labelled a-f, from LB + ampicillin 100μl plate, and one colony, labelled x, from LB + ampicillin 10μl plate.
- We performed colony PCR according to the following protocol, using BBa_I13520_pSB1A3 plasmid as a positive control. We used standard [http://partsregistry.org/Part:BBa_G00101 VR] and [http://partsregistry.org/Part:BBa_G00100 VF2] primers, as indicated on the diagrams below:
| Expected lengths of products: | |
| Positive control | ~2400 bp |
| GA1 insert | ~2800 bp |
| GA3 insert | ~2800 bp |
- As we can see from the picture of the agarose gel, c-f and x colonies carried correctly assembled plasmids. We decided to prepare glycerol stocks of these successful transformants.
Diagnostics
In addition, we performed a series of tests to identify the cause of the low efficiency of transformation. The proposed sources of error included:
- Low efficiency of DNA gel extraction
- Unsuccessful Gibson Assembly
- Low viability of competent E.coli cells
In order to check if gel extraction of DNA was successful, we ran a 1% agarose gel with samples of purified products of the initial PCR reactions. Although we could see distinct, fairly thick bands on the first gels, from which components of Gibson constructs were purified, bands on the diagnostic gel are very faint, even missing in some lanes. This indicates that the yield of our extraction procedure was very low and probably that was the main reason why the performed transformation was fairly unsuccessful.
Therefore, we decided to repeat PCR reactions, this time at higher 50μl volume, and try different conditions of the gel extraction procedure.
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 performed PCR using Phusion Hot Start DNA Polymerase in 50 μl reaction volume.
- The time profile used in the PCR machine was the following, the same as the one used before:
| Hold | 95°C | 2 min | |
| Cycling | Denaturing | 95°C | 10 s |
| Annealing | 55°C | 20 s | |
| Elongation | 72°C | 150 s |
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.
- In most cases position of a band matches the expected length of DNA fragment. The only exception are again 4-5kb GA1-2 and GA3-2 products, although the predicted length is 3.5kb.
- This time we obtained single bands on lanes with amplified reflectin, whereas we observed some additional bands on almost all lanes with amplified backbones of plasmids to be constructed. Most probably these were products of non-specific annealing, which we did not detect on the first gel due to low concentration of these additional products in 25μl reaction. Moreover, these differences emphasize the fact that the amount of non-specific products of DNA amplification can vary greatly between reactions depending on the timing of random mis-annealing events.
- 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.
We also ran a gel with products of extraction to check how efficient the procedure was.
- We obtained clear distinct bands on each lane loaded with components of GA5 and GA6 Gibson Assembly constructs.
Gibson Assembly
We conducted Gibson Assembly of GA1, GA2, GA3, GA4, GA5 and GA6 constructs according to the protocol. The total reaction volume was 20μl.
Transformation
We transformed competent E.coli cells according to the following protocol. We cultured each class of transformants on four different plates.
| GA1, GA3 and positive control | GA2, GA4, GA5 and GA6 | |
| Plate 1 non-induced (10μl of cell suspension) | LB + ampicillin | LB + kanamycin |
| Plate 2 non-induced (100μl of cell suspension) | LB + ampicillin | LB + kanamycin |
| Plate 3 induced (10μl of cell suspension) | LB + ampicillin + arabinose | LB + kanamycin + arabinose |
| Plate 4 induced (100μl of cell suspension) | LB + ampicillin + arabinose | LB + kanamycin + arabinose |
As a positive control, we transformed cells with BBa_13520_pSB1A3 plasmid, expecting to detect RFP fluorescence on induced plates. As a negative control, we plated non-transformed heat-shocked competent cells on LB + kanamycin and LB+ampicillin plates.
After overnight incubation at 37°C we examined plates under a fluorescence microscope to check if the cell had been also transformed with the template DNA used in the initial PCR reactions. The table presents expected phenotype of cells transformed with the correct Gibson Assembly construct or two template DNA plasmids.
| Type of plasmid | Expected phenotype of transformed colonies | |
| LB + Antibiotic | LB + Antibiotic + Arabinose | |
| Reflectin A1 expression plasmid | No fluorescence | No fluorescence |
| I13520:pSB1A3 plasmid | No fluorescence | RFP red fluorescence |
| J69511:pSB3K3 plasmid | GFP green fluorescence | GFP green fluorescence |
We observed colonies on all plates except for the negative control:
| positive control | red fluorescence on LB + ampicillin + arabinose plate (as expected) |
| GA1 | no fluorescence detected |
| GA2 | green fluorescence of some colonies on LB + kanamycin + arabinose plates (both 100μl and 10μl) |
| GA3 | no fluorescence detected |
| GA4 | green fluorescence of some colonies on both induced and non-induced plates |
| GA5 | green fluorescence of some colonies on both induced and non-induced plates |
| GA6 | green fluorescence of some colonies on both induced and non-induced plates |
Results
What next?
