Team:WLC-Milwaukee/Experiments
Wet Lab
Our Wet Lab efforts focused on first designing our parts in such a way as to test new versions of past WLC-iGEM binding proteins for optimization and use in our test kit. Our work then turned to successfully sub-cloning our new parts (BBa_K2589000, BBa_K2589002 ) from the pETail4 plasmid containing the lambda phage genome into both pTrc99a and pSB1C3 along with improving a past part BBa_K2452002 to create the composite part BBa_K2589001. Our final focus in WetLab was to purify the proteins from our new parts using Nickel column purification (as most of our parts contain a His tag element) and then confirming expression or protein presence.
Part Design
Our Wet Lab team and Dr. Werner met early in the year to discuss the full spectrum of possible parts we would attempt to create and submit, and as with many iGEM teams we set our goals a bit too high, hoping to create a series of fusion proteins with which to better analyze our test kit on top of the basic parts needed for our system. We analyzed the sequence of the pTrc99a plasmid and decided to use the trc-promoter BBa_K2042004 for induction of all of our lambda phage tail protein parts with IPTG. Because the part sample for this promoter has not been released, primers had to be designed to input our parts into the pTrc99a backbone and then further clone our parts into pSB1C3 with the trc-promoter from the pTrc99a backbone.
The basic parts we designed at the beginning of our project include BBa_K2589000 and BBa_K2589002, and an improved composite part BBa_K2589001 consisting of BBa_K2452002 and BBa_K2042004. Dr. Werner helped us design our primers in a major way ensuring our sequences were accurate and should work as expected. We did run into some problems with low gene amplification during PCR and ended up increasing the size of our original primers by 1bp.
Cloning
We followed standard procedures for the cloning of our parts, first amplifying our genes of interest by PCR, digesting and ligating our parts into our backbone(s), and transformation via electroporation into electrocompetent lab strain E. coli. Successful transformants underwent initial screening through diagnostic restriction enzyme digests to confirm the presence of our plasmid backbones and appropriate genes.
When diagnostic digests showed high potential for a successful clone, samples were sent for sequencing to (in most cases) confirm the presence and accurate sequence of our parts. We ran into numerous issues during cloning that slowed the progression of our lab work and project including poor gene amplification during PCR which was inevitably solved using slightly longer primers and a longer annealing time. Additionally, we had some unexpected initial sequencing data that confounded the situation, however, after additional screening we believe our part sequences to be accurate and complete. See Results for data and the results of our cloning
Protein Expression and Purification
After confirmation of successful cloning of our parts, we began the process of protein purification. Strains with successful clones were cultured and then diluted and induced using IPTG for maximal protein expression. Our proteins were purified using a Nickel column purification kit/system from Qiagen (Ni-NTA Fast Start Kit) after which a series of tests (some successful some not) were performed in an attempt to confirm the presence and functionality of our purified proteins, including PAGE gels with Coomassie Staining. We then proceeded to conjugate Horseradish peroxidase to our protein samples and performed His-tag specific Western Blotting and chemiluminescent tests (using our parts combined with various diluted cultures of E.coli and exposed to a chemiluminescent substrate cleaved by HRP) to confirm the purification, conjugation of HRP to, and binding of, our proteins to E.coli. To find more information regarding our testing please see Results and System Design.
