Team:Cambridge/Experiments/Synthetic Reflectin PCR and Construction of GA1 to 6
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These constructs were grown up in E.Coli and verified by [[Team:Cambridge/Protocols/Colony_PCR | colony PCR]]. The plasmids were then [[Team:Cambridge/Protocols/Mini_Prep | miniprepped ]] and stored at -80°C. | These constructs were grown up in E.Coli and verified by [[Team:Cambridge/Protocols/Colony_PCR | colony PCR]]. The plasmids were then [[Team:Cambridge/Protocols/Mini_Prep | miniprepped ]] and stored at -80°C. | ||
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+ | {| | ||
+ | |Name | ||
+ | |Description | ||
+ | |- | ||
+ | |GA1 | ||
+ | |Reflectin A1 | ||
+ | |on PSB1A2 (high copy) | ||
+ | |pBAD promoter + B0015 terminator | ||
+ | |- | ||
+ | |GA2 | ||
+ | |Reflectin A1 | ||
+ | |on PSB3K3 (low copy) | ||
+ | |pBAD promoter + B0015 terminator | ||
+ | |- | ||
+ | |GA3 | ||
+ | |Reflectin A1 + HIS tag | ||
+ | |on PSB1A2 (high copy) | ||
+ | |pBAD promoter + B0015 terminator | ||
+ | |- | ||
+ | |GA4 | ||
+ | |Reflectin A1 + HIS tag | ||
+ | |on PSB3K3 (low copy) | ||
+ | |pBad promoter + B0015 terminator | ||
+ | |- | ||
+ | |GA5 | ||
+ | |Reflectin A2 | ||
+ | |on PSB3K3 (low copy) | ||
+ | |pBAD promoter + B0015 terminator | ||
+ | |- | ||
+ | |GA6 | ||
+ | |Reflectin 1B | ||
+ | |on PSB3K3 (low copy) | ||
+ | |pBAD promoter + B0015 terminator | ||
===Detail=== | ===Detail=== |
Revision as of 15:29, 17 September 2011
Contents |
Synthetic Gene Amplification & Plasmid Construction
Synopsis
The synthetic genes were extracted from the filter paper on which they were sent, and amplified by PCR.
Six plasmids were then constructed, named GA 1-6, as shown below. Each puts reflectin expression under the control of pBAD promoter, an arabinose inducible promoter. HIS tags were included on some of the constructs in order to purify the protein for in vitro work. In addition, we put both reflectin A2 and 1B in a low expression plasmid.
Name | Description | |
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GA1 | Reflectin A1 | on PSB1A2 (high copy), pbad promoter + B0015 terminator |
GA2 | Reflectin A1 | on PSB3K3 (low copy), pbad promoter + B0015 terminator |
GA3 | Reflectin A1 + HIS tag | on PSB1A2 (high copy), pbad promoter + B0015 terminator |
GA4 | Reflectin A1 + HIS tag | on PSB3K3 (low copy), pbad promoter + B0015 terminator |
GA5 | Reflectin A2 | on PSB3K3 (low copy), pbad promoter + B0015 terminator |
GA6 | Reflectin 1B | on PSB3K3 (low copy), pbad promoter + B0015 terminator |
These constructs were grown up in E.Coli and verified by colony PCR. The plasmids were then miniprepped and stored at -80°C.
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GA1 | Reflectin A1 | on PSB1A2 (high copy) | pBAD promoter + B0015 terminator | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GA2 | Reflectin A1 | on PSB3K3 (low copy) | pBAD promoter + B0015 terminator | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GA3 | Reflectin A1 + HIS tag | on PSB1A2 (high copy) | pBAD promoter + B0015 terminator | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GA4 | Reflectin A1 + HIS tag | on PSB3K3 (low copy) | pBad promoter + B0015 terminator | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GA5 | Reflectin A2 | on PSB3K3 (low copy) | pBAD promoter + B0015 terminator | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GA6 | Reflectin 1B | on PSB3K3 (low copy) | pBAD promoter + B0015 terminator
DetailExtraction & AmplificationThe sample plasmids containing the reflectin gene are first extracted from filter paper, and then used in the transformation of competent E. coli cells. A standard miniprep can then be used to recover the plasmid DNA from the bacteria. The purpose of this procedure is to amplify and store the plasmids' DNA.
Miniprep extraction
Healthy colonies of transformed E. coli were observed after incubation in LB agar, indicating the success of the amplification procedure. Construct DesignIn order to work with Reflectin in vivo and in vitro we needed to create DNA constructs that would allow us to control expression level and to tag the protein for purification. The first two constructs we made, GA1, GA2, put reflectin expression under the control of pBAD an arabinose controlled promoter (one on high copy pSB1A2 plasmid, one on the low copy pSB3K3 plasmid), whilst GA3 and GA4 also did this, they incorporated a His-tag to enable purification at a later stage.
Assembly of GA1, GA2, GA3 and GA4 - First AttemptPCRIn the first round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3 and GA4 constructs.
We decided to use the 55°C annealing temperature, although the predicted temperature for most primers is 5-10°C higher, because of a low annealing temperature of the VF2 primer.
For the gel extraction of DNA we followed the protocol, assuming that one slice of gel is 100μl. Gibson AssemblyWe 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:
TransformationWe transformed competent E.coli cells according to the following protocol. We cultured each class of transformants on four different plates.
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.
Colony PCRWe decided to conduct colony PCR of E.coli transformed with GA1 and GA3 constructs in order to confirm successful assembly of the plasmids.
pSB1AK3 or pSB1A3 backbone?As a side experiment, we decided to determine whether constructs with pSB1A3 backbone also carry a gene for kanamycin resistance. When preparing glycerol stocks of successful transformants, we cultured GA1x and GA3c bacteria in LB + kanamycin liquid medium in addition to LB + ampicillin medium. After 4 hour incubation at 37° the turbidity of both cultures was the same. DiagnosticsIn addition, we performed a series of tests to identify the cause of the low efficiency of transformation. The proposed sources of error included:
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 of GA1, GA2, GA3, GA4, GA5 and GA6 - second attemptPCRIn 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.
We also ran a gel with products of extraction to check how efficient the procedure was.
Gibson AssemblyWe conducted Gibson Assembly of GA1, GA2, GA3, GA4, GA5 and GA6 constructs according to the protocol. The total reaction volume was 20μl. TransformationWe transformed competent E.coli cells according to the following protocol. We cultured each class of transformants on four different plates.
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.
Colony PCRWe conducted colony PCR of E.coli transformed with GA1 - GA6 constructs in order to confirm successful assembly of these plasmids.
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