Contributors: Chester
This entry backfills dry-lab planning work that actually spanned an extended period (predating the wet-lab epPCR runs from 30 June onward) but has no recorded date — filed under the compilation date instead.
Plasmid reconstruction
The iGEM Registry provides the CxnA insert and the pSB1C3 backbone as separate parts, so reconstructing the theoretical full plasmid required computationally joining them at the correct BioBrick insertion site.
In pSB1C3, the relevant restriction sites run EcoRI → XbaI → SpeI → PstI left to right, with the CxnA insert expected between XbaI and SpeI. Because BioBrick parts are modular by design, the insert drops into that defined region and the restriction-site junctions can be treated as standardised boundaries for reconstructing the full sequence.
If the original construct (Chris French) was assembled by ligation rather than Gibson or Golden Gate, scar sequences would be expected at the ligation points — but any effect from that is unlikely to matter, since the ligation points fall outside the coding regions.
Sequencing verification
Sequenced both available CxnA plasmid variants — plain CxnA and His-tagged CxnA — using existing forward/reverse primers inherited from previous teams, as a preliminary check against the reconstructed reference sequence. Four reads: Forward 1 / Reverse 1 (CxnA-His), Forward 2 / Reverse 2 (CxnA).
Two problems surfaced:
- Coverage gap. Both ends sequenced cleanly but left a large unread gap in the middle — the insert was too long for the reads to span in one pass (each primer reliably covers roughly 1000 bp before accuracy drops). Fix: designed two additional forward primers, Lone-Forward 1 (just before the gap) and Lone-Forward 2 (inside the gap), sized to close it between them. No new reverse primers were needed — the forward reads plus reverse-complementing the template strand give equivalent information.
- His-tag artefact. A large low-confidence (flagged) region appeared in Forward 2 only. Traced to the His-tag: pair 1 (CxnA-His) had template to sequence through it, pair 2 (untagged CxnA) did not, producing a misleading gap when the two pairs were aligned against each other.
[Attachment: sequencing trace / chromatogram images not supplied]
Gibson assembly primer design
Chose Gibson assembly over designed restriction sites because pSB1C3 + CxnA already carry a diverse set of restriction sites, and a unique site would be needed at every ligation point — none were available among the enzymes in stock. Gibson ligates scarlessly instead, using overlapping primer regions plus an exonuclease-based kit (per Heather) to generate compatible overhangs.
Two separate mutagenesis targets need their own primer pairs, both sharing the same start cutoff (beginning of the CxnA coding sequence, excluding the upstream promoter/RBS):
| Target | Project | Start cutoff | End cutoff | Primer pair |
|---|---|---|---|---|
| Whole-CxnA epPCR (general optimisation) | B | Start of CxnA | CxnA–backbone junction | For-GibStart + For-GibEnd / Rev-GibEnd |
| Cex/GH10-only epPCR (beta-glucosidase evolution) | A | Start of CxnA | End of the Cex exoglucanase region (mid-sequence) | For-GibStart + For-GibMid / Rev-GibMid |
GC content: C. fimi is an Actinomycete with a high-GC genome, making the usual 40–60% primer GC target harder to hit. GibStart/GibEnd pairs came in just under 60%. GibMid was initially 75% and had to be shifted slightly further into the Cex region — without reaching into the catalytic motifs — bringing it down to 65%. Lone-Forward 1 and 2 both slightly exceed 60%; Heather and Michael noted this is common (and usually still works) for Actinomycete templates.
Status
Primer design and sequencing-gap resolution are done. Actual Gibson assembly and library construction are tracked under the wet-lab epPCR entries (30 June onward) — this entry covers only the dry-lab design work that preceded them.