Difference between revisions of "Team:WLC-Milwaukee/Demonstrate"
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| + | <p>Once the expression was verified, our team decided the best purification method of the tail tip proteins would be sub-cloning and his-tagging genes off of this plasmid. To improve on a previous part, we attached a His-tag (part BBa_K112703) to various sizes and ends of the tail gene. We then inserted the genes into ptrc99A backbones. This vector allows for inducible expression with IPTG. We used the E. coli strain DH5 alpha for expression of the genes. Using a nickel column, we attempted native purification of an N-terminus, shortened version of the J-chain tail tip protein. The SDS-PAGE done after this shows that the nickel column did not bind the protein. This may be because the his-tag became buried in the protein after folding. To verify the his-tag was present, we did a Western blot using a his-tag specific antibody.</p> | ||
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Revision as of 00:14, 29 October 2017
Results
Test Kit Assessment
To begin designing and experimenting with a new device for E. coli detection, the WLC iGEM team wanted to test already marketed water test kits. Using both laboratory strain and wild type E. coli, the WLC iGEM team evaluated the reliability of six test kits while assessing other features of the kits. We found that while the kits were relatively accurate, they could not detect lab strain E. coli, and most required 48 hours for results. Below is a table summarizing our findings:
pETail4 and Lambda phage tail proteins
To continue with our new device, we obtained the pETail4 plasmid, which contains the genes for the Lambda Phage-tail proteins. The pETail4 plasmid has been used in past research to produce Lambda phage tails for determining the distribution of the LamB protein on the outer membrane of E. coli. The tail itself is composed of several smaller proteins. It has been proposed that the portion of the tail specific to LamB is the J-chain. Thus, we wanted to isolate this portion, or at least the tip of this portion. We verified the expression of the J-chain with an SDS-PAGE (below)
Sub-cloning and His-tag purification
Once the expression was verified, our team decided the best purification method of the tail tip proteins would be sub-cloning and his-tagging genes off of this plasmid. To improve on a previous part, we attached a His-tag (part BBa_K112703) to various sizes and ends of the tail gene. We then inserted the genes into ptrc99A backbones. This vector allows for inducible expression with IPTG. We used the E. coli strain DH5 alpha for expression of the genes. Using a nickel column, we attempted native purification of an N-terminus, shortened version of the J-chain tail tip protein. The SDS-PAGE done after this shows that the nickel column did not bind the protein. This may be because the his-tag became buried in the protein after folding. To verify the his-tag was present, we did a Western blot using a his-tag specific antibody.
