Team:uOttawa/ResultsTemp
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The ~4 KB band shown in figure 5 was cut out of the gel and transformed into yeast using standard transformation protocols. Figure 6 shows a picture of the transformation plate, red colonies indicate positive transform ants. | The ~4 KB band shown in figure 5 was cut out of the gel and transformed into yeast using standard transformation protocols. Figure 6 shows a picture of the transformation plate, red colonies indicate positive transform ants. | ||
===Results=== | ===Results=== | ||
- | [[File:Mason7.png|center| | + | [[File:Mason7.png|center|250px|thumb|'''Figure 6.''' Yeast cells growing in the presence of G418 after transformation with the construct. Red colonies indicate that the construct integrated at the correct genomic locus. There are 31 red colonies and 24 white colonies.]] |
16 red colonies form the plate shown in figure 6 were restreaked onto SM – Ura plates, and all of them grew, the wild type BY4742 strain did not grow. | 16 red colonies form the plate shown in figure 6 were restreaked onto SM – Ura plates, and all of them grew, the wild type BY4742 strain did not grow. | ||
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===Benchmarking=== | ===Benchmarking=== | ||
Our construct was amplified out of each of the 16 colonies and was sequenced. Out of the 40 000 bp that were sequenced, there were only 7 definitive mutations identified. Therefore we have benchmarked out method as having an error rate of 0.0175 errors per nucleotide. Unfortunately many of these errors occurred at the junction of the Ura3-GFP fusion protein causing the GFP to be out of frame. This error occurred early on in the procedure during heterodimer formation and propagated throughout the assembly. That is why all the colonies grew in –Ura but only 6 expressed GFP. Most likely this problem is related to the ligase rather than multiple rounds of PCR. | Our construct was amplified out of each of the 16 colonies and was sequenced. Out of the 40 000 bp that were sequenced, there were only 7 definitive mutations identified. Therefore we have benchmarked out method as having an error rate of 0.0175 errors per nucleotide. Unfortunately many of these errors occurred at the junction of the Ura3-GFP fusion protein causing the GFP to be out of frame. This error occurred early on in the procedure during heterodimer formation and propagated throughout the assembly. That is why all the colonies grew in –Ura but only 6 expressed GFP. Most likely this problem is related to the ligase rather than multiple rounds of PCR. | ||
- | === | + | ===Summary=== |
- | + | We developed a new DNA assembly protocol called BrickMason assembly. We assembled 6 pieces in one day's work and showed that the assembled constructs functioned as expected in yeast. The yeast cells were extensively sequenced and we found that the method had an error rate of 0.0175 errors per nucleotide. | |
+ | The same cloning protocol would have taken 9 days to complete using traditional cloning methods. The whole procedure can be completed with standard lab equipment and reagents. Users who will not be using E. coli as the final destination for their constructs do not need to use E. coli at all, they can instead transform the final construct directly into the organism of interest. We hope that BrickMason makes you enjoy cloning as much as we do! |
Latest revision as of 02:43, 29 September 2011
Contents |
BrickMason Results
The construct
In order to test the BrickMason assembly method, we decided to build the following construct:
When transformed into yeast this construct should knock out the ADE2 gene, causing the cell to turn red. Cells that have successfully taken up the construct should be resistant to the G418 drug (conferred by KanMx) and should express the Ura3-GFP fusion protein. They should therefore also grow on Ura- plates, and express GFP.
The assembly
Constructs 1-6 were PCRed using biobrick primers and then cut out of a gel, This is shown below in figure 2. The ladder used in all of our gels is the NEB 2-Log DNA Ladder which can be found at: http://www.neb.com/nebecomm/products/productN3200.asp
These pieces were then used to create overlapping heterodimers as explained in the BrickMason animation and description. Part A of the heterodimer was digested with SpeI, and part B was digested with XbaI, part B was then dephosphorylated. Part A and B were ligated together, and were then digested with XbaI and SpeI to get rid of homodimers. This reaction mixture was used as a template in the a PCR reaction using the forward BioBrick primr for part A and the reverse BioBrick primer for part B. The heterodimers used in this assembly (12,23,34,45,56) are shown in figure 4.
Each heterodimer was cut out of the gel in figure 4, gel purified, and digested with XbaI and SpeI. These heterdimers were then combined in one final PCR reaction to obtain the final construct shown in figure 5.
The ~4 KB band shown in figure 5 was cut out of the gel and transformed into yeast using standard transformation protocols. Figure 6 shows a picture of the transformation plate, red colonies indicate positive transform ants.
Results
16 red colonies form the plate shown in figure 6 were restreaked onto SM – Ura plates, and all of them grew, the wild type BY4742 strain did not grow.
These colonies were then inoculated into liquid culture and grown overnight and measure on the flow cytometer. Figure 8 shows that 6 of the 16 colonies expressed GFP.
Benchmarking
Our construct was amplified out of each of the 16 colonies and was sequenced. Out of the 40 000 bp that were sequenced, there were only 7 definitive mutations identified. Therefore we have benchmarked out method as having an error rate of 0.0175 errors per nucleotide. Unfortunately many of these errors occurred at the junction of the Ura3-GFP fusion protein causing the GFP to be out of frame. This error occurred early on in the procedure during heterodimer formation and propagated throughout the assembly. That is why all the colonies grew in –Ura but only 6 expressed GFP. Most likely this problem is related to the ligase rather than multiple rounds of PCR.
Summary
We developed a new DNA assembly protocol called BrickMason assembly. We assembled 6 pieces in one day's work and showed that the assembled constructs functioned as expected in yeast. The yeast cells were extensively sequenced and we found that the method had an error rate of 0.0175 errors per nucleotide. The same cloning protocol would have taken 9 days to complete using traditional cloning methods. The whole procedure can be completed with standard lab equipment and reagents. Users who will not be using E. coli as the final destination for their constructs do not need to use E. coli at all, they can instead transform the final construct directly into the organism of interest. We hope that BrickMason makes you enjoy cloning as much as we do!