Difference between revisions of "Team:Austin UTexas/Results/Parts"
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| − | <p> Before the plasmids could be assembled, the contributing parts must be isolated, affixed with restriction sites and customized overhangs, and inserted into a backbone so the parts could be stabilized and stored in a part plasmid. In a Golden Gate Assembly cloning reaction, the DNA sequence for the plasmid part is inserted into a plasmid containing a gene for green fluorescent protein production (GFP), on either side of which, are sites where the BsmBI enzyme cuts. The GFP gene is replaced by the plasmid part, so colonies that do not fluoresce green under UV light are sustaining the part plasmid. <b>Over the course of this project we have made a total of | + | <p> Before the plasmids could be assembled, the contributing parts must be isolated, affixed with restriction sites and customized overhangs, and inserted into a backbone so the parts could be stabilized and stored in a part plasmid. In a Golden Gate Assembly cloning reaction, the DNA sequence for the plasmid part is inserted into a plasmid containing a gene for green fluorescent protein production (GFP), on either side of which, are sites where the BsmBI enzyme cuts. The GFP gene is replaced by the plasmid part, so colonies that do not fluoresce green under UV light are sustaining the part plasmid. <b>Over the course of this project we have made a total of 48 parts.</b> For coding sequences and promoter/RBS parts, not all of the varieties of the part will be used in the assemblies. Rather, they are meant to provide a resource of alternative parts to a researcher constructing their own plasmid from the kit. For barcodes and 1-5 bridges, each must be unique to the origin in their plasmid because they are meant to identify which origin the plasmid contains. Having many varieties of antibiotic resistance and origin of replication strengthens the kit, as it allows researchers to rapidly test more origins at a time, in bacteria that may be naturally resistant to some antibiotics. </p> |
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| − | <img | + | <img src="https://static.igem.org/mediawiki/2018/f/f9/T--Austin_UTexas--partplasmidp8.JPG" style="width:400px";> |
| − | <figcaption><b>Figure 1.</b> | + | <figcaption><b>Figure 1.</b> P8 promoter part plasmid E. coli transformation, compared to control transformations with entry vector pYTK001. Under UV illumination, transformants containing the correctly assembled part plasmids were non-fluorescent while negative transformants appeared fluorescent like colonies on the control plates. Picture taken by Andrew Ly.</figcaption> |
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| + | <img src="https://static.igem.org/mediawiki/2018/c/cf/T--Austin_UTexas--improvedgrayparts.png" style="width:600px";> | ||
| + | <figcaption><b>Figure 2.</b> A list of every part plasmid included in the kit.</figcaption> | ||
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Latest revision as of 23:29, 17 October 2018
Part Plasmids
Before the plasmids could be assembled, the contributing parts must be isolated, affixed with restriction sites and customized overhangs, and inserted into a backbone so the parts could be stabilized and stored in a part plasmid. In a Golden Gate Assembly cloning reaction, the DNA sequence for the plasmid part is inserted into a plasmid containing a gene for green fluorescent protein production (GFP), on either side of which, are sites where the BsmBI enzyme cuts. The GFP gene is replaced by the plasmid part, so colonies that do not fluoresce green under UV light are sustaining the part plasmid. Over the course of this project we have made a total of 48 parts. For coding sequences and promoter/RBS parts, not all of the varieties of the part will be used in the assemblies. Rather, they are meant to provide a resource of alternative parts to a researcher constructing their own plasmid from the kit. For barcodes and 1-5 bridges, each must be unique to the origin in their plasmid because they are meant to identify which origin the plasmid contains. Having many varieties of antibiotic resistance and origin of replication strengthens the kit, as it allows researchers to rapidly test more origins at a time, in bacteria that may be naturally resistant to some antibiotics.
