Difference between revisions of "Team:iTesla-SoundBio/Parts"

This would be the coding sequence for Limulus Clotting Factor C. To create this, we would first acquire the amino acid sequence from UniProt (UniProtKB - P28175 (LFC_TACTR)), then codon optimize for B. subtilis using JCat and add the BioBrick prefix and suffix. The Factor C sequence is just over the IDT length limit for free synthesis--the limit is 3kb, while the sequence was 3,133 bases long--so we would need to synthesize it as two separate parts. We would independently digest and ligate these fragments into two separate plasmid backbones, then PCR the plasmids, digest the fragments out, ligate them together, and finally ligate them into the backbone pSB1C3. To design those fragments, we would have them overlap at a BioBrick-compatible restriction site in the middle of the Factor C sequence. This would be used to ligate the fragments together once we had purified samples of each.

Each Factor C fragment would also be flanked by a BioBrick prefix and suffix, and the theoretical completed Factor C gene would be flanked by the restriction sites BamHI and SacII. These restriction sites are part of the BioBrick prefix and suffix and would be useful for future digestion and ligation into an expression vector. We would then separately digest Fragment 1 and our BioBricking vector (pSB1C3) using restriction enzymes PstI and EcoRI, then ligate those two components together and transform that Fragment 1 + pSB1C3 complex into E. coli. After growing copies of that plasmid with Fragment 1 inside, we would miniprep successful colonies and sequence them to ensure success.

Afterwards, we would digest the Fragment 1 + pSB1C3 complex with the BglII and PstI restriction enzymes, making an opening that would allow Fragment 2 to ligate with the appropriate side of Fragment 1. After digesting Fragment 2 with the same restriction enzymes, we would ligate and transform those components into E. coli. If all went according to plan, the resulting colony would contain the pSB1C3 + Fragment 1 + Fragment 2 complex and thus the completed BioBrick.

Our attempts to carry out this procedure were foiled by our repeated failure to successfully execute digests. If we were to attempt this again, we would design a geneblock comprising the entire Factor C sequence save ~70 bases on each end, then use extra-long primers to add in the rest. This would require fewer total steps and thus have a lower chance of failure.

This would be a BioBrick-compatible version of the Bacillus integration vector pAX01. Unlike BBa_J179001--to which it is otherwise identical--it would include the xylose-inducible xylR+PxylA repressor-promoter system specified by the part BBa_K733002. This would allow transcriptional regulation of the cloned gene through the addition and removal of xylose.

After getting the pAX01 plasmid from BGSC, we would need to make numerous modifications to make it BioBrick-compatible, including the insertion of the BioBrick prefix and suffix at the MCS. The first step, however, would be confirming the sequence of the plasmid in our possession. We would use twelve sequencing primers to do this (see Table 1). Once the sequence of the plasmid was confirmed, we would carry out a series of steps that, if completed correctly, would result in the successful synthesis of the pAX01 BioBrick.

The first step would be to swap out the xylose cassette in pAX01 with BBa_K733002, removing a few BioBrick-incompatible restriction sites. This would require FastCloning: we would begin by running PCRs of BBa_K733002 and pAX01 such that the product of pAX01 would exclude its xylose cassette (using the FastClone primers in Table 1), then we would digest the PCR products with DpnI, transform them into E. coli, and miniprep the resulting plasmids. We would then send these off for sequencing; if the FastClone were successful, its product would be pAX01 with its xylose cassette region replaced by BBa_K733002.

Next, we would need to perform site-directed mutagenesis on pAX01’s AmpR region by running a PCR with the primers pAX01 Point Mut Fwd and pAX01 Point Mut Rev (see Table 1). We would digest the PCR products with DpnI, transform them into E. coli, then miniprep and sequence the resulting plasmids.

We would next run two deletions, each using the same protocol as that of the AmpR mutagenesis except for the primers (see Table 1). Finally, we would run an insertion, again using the same protocol except for the primers (see Table 1).

We began by trying to FastClone the xylose cassette BBa_K733002 into pAX01, but our PCRs and transformations consistently failed and we were unable to construct the BioBrick. This may have been a result of poor primer design, incorrect annealing temperatures, incorrect transformation procedures, and/or human error in carrying out the procedures. If we were to do this again, we would document our labs much more thoroughly and possibly try a technique other than FastClone for the BBa_K733002 insertion.

Table 1: