Team:iTesla-SoundBio/Project/results
Results
Synthesis of a Biobrick-compatible Factor C Plasmid in E. coli
Figure 1.2
Well 1, empty; Well 2, ladder; Well 3, Fragment 1 PCR; Well 4, Fragment Well 1, ladder; Well 2, empty; Well 3, Fragment 1 PCR; Well 4, empty; Well 5, Fragment 1 PCR. There was amplification, but the DNA that appeared in the gel was less than 1 kb, which is much shorter than the expected length (~ 1.5 kb). There were also smears.
One of our biggest issues was that few PCRs of our Factor C fragments were successful. Results often showed nonspecific bands,smears or bands that were shorter than the desired length (as shown in Figures 1.1 and 1.2). We realized late in the year that our biobricking primers were poorly designed and did not have enough bases matching our fragments, so they were annealing incorrectly. We tried re-designing primers and adjusting the annealing temperature and extension times, but we still could not get the PCRs working efficiently. We still attempted to purify some PCRs and use them in cloning, but the poor result of our PCRs was one of the biggest factors in our failed transformations which are discussed below.
Figure 2.1
Row 1, no DNA negative control; Row 2, positive control; Row 3, No insert; Row 4, experimental (should have had factor C fragment, no mRFP). The red colonies on the no insert plate indicate that there was digested plasmid that ligated back togetherand the mRFP was not removed and replaced with Factor C successfully.
Figure 2.2
Top: No insert control; Bottom: experimental.
Figure 2.3
Digest of BBa_J04450 (mRFP) + pSB1C3. The upper bands (pSB1C3) were gel extracted and used for ligation with the factor C fragments.
Our transformations were consistently unsuccessful. We kept having the same issue: the presence of red colonies. The gel above is an example of the results from our restriction digests of BBa_J04450 with PstI and EcoRI; they consistently worked and the mRFP was cut out of the pSB1C3 vector, giving 2 distinct bands. We attempted gel extractions of pSB1C3 (upper band on figure # 2.3, ~ 2 kb), but our bacteria still expressed mRFP after ligation and transformation. There were some white colonies, but we believe they were two vectors ligated together and did not contain our desired factor C fragment. Since we continued having this issue, we attempted to transform E. coli using an alternative method called FastCloning [1].
Figure 3.1
Figure 3.2
In the FastClone method, the vector and insert are designed to have overlapping regions at the site of insertion. The biobrick prefix and suffix can serve as this overlap. First, the vector (pSB1C3) and insert (Factor C fragment) are amplified using PCR with Phusion or another high fidelity polymerase. During PCR, Phusion can exhibit 3’ exonuclease activity that creates sticky ends at each 3’ end of the amplified strand of DNA. The PCR reactions of the vector and insert are then mixed together and digested with DpnI, a restriction enzyme that only cuts methylated DNA; in other words, DpnI will cut the template plasmid DNA (provided it was derived from an organism that methylates DNA like E. coli) used in PCR to prevent it from being expressed by bacteria. The overlapping regions allow the insert and vector to anneal when mixed and form a nicked plasmid which is then ligated in vivo.
Before working with pAX01 or Factor C, we attempted a practice transformation using the FastClone method. We attempted to insert a CFP gene into the pSB1C3 backbone, but as seen in Figure 3 above, none of the colonies that grew on the plates expressed CFP, and some expressed mRFP as in the earlier transformations. We also had fewer transformants. We suspect that the DpnI digest did not destroy all of the template DNA, and the only DNA that the bacteria were transformed with was the original BBa_J04450 plasmid or two pSB1C3 vectors that annealed together. The purpose of shifting our focus to FastCloning was to quickly and easily clone the full Factor C gene into E. coli, but it was ultimately unsuccessful.
Synthesis of BioBrick-compatible pAX01
We also hoped to make our pAX01 plasmid biobrick compatible. There is currently a version of the pAX01 plasmid in the iGEM registry; however, our goal was to submit a version of the plasmid that also contained the xylose-inducible expression system. We designed several pairs of primers for site-directed mutagenesis that would remove all of the illegal cut sites. The proposed process designed by our team is a series of PCRs and transformations that removes and inserts new pieces until we attain our desired biobrick, but there was difficulty in primer design and we had little success with our PCR experiments. There were ultimately no successful transformations, and we were unable to create the biobrick part of pAX01 that we designed.
Synthesis of Factor C-producing B. subtilis
In parallel to the attempted biobricking work, we worked towards transforming B. subtilis 168 with our chosen integration vector, pAX01. Integration with pAX01 would be a necessary part of expressing Factor C, so it was important to confirm that it worked properly even though we were unable to successfully clone Factor C. Upon successful integration of pAX01 into the genome, B. subtilis will become resistant to erythromycin. Based on this antibiotic selection, we had some apparently successful transformations, albeit with low efficiency and after many attempts.
We then attempted a colony PCR on our transformants as a preliminary method of confirming successful integration. We designed primers to amplify the integrated portion of DNA between portions of the B. subtilis 168 genome.
Our colony PCRs gave us mixed results. Sometimes the PCR experiments resulted in no apparent amplification and there was only genomic DNA stuck in the wells (figure #). In other attempts, one primer pair gave us the desired results but also showed nonspecific bands from other parts of the B. subtilis genome. Since many colony PCRs of our transformations did not work at all, we predicted that what we thought was transformed B. subtilis was not in fact B. subtilis and was instead contamination from another organism. The colonies of the transformations grew unusually compared to our regular B. subtilis, and we had several issues with contamination of buffers when attempting transformations. So, in addition to doing colony PCRs designed to confirm successful integration, we also amplified the 16s region of our bacteria so that we could then sequence it and confirm the organism’s identity. We also redesigned our primers for integration confirmation hoping to get fewer nonspecific bands and more accurate results.