Team:Hong Kong HKUST/pneumosensor/results/module one

The two-component regulatory system in S. pneumoniae, consisting of the receptor ComD and its response regulator ComE was to be used in detecting the autoinducer molecule, competence-stimulating peptide (CSP) and so detect S. pneumoniae populations correspondingly.

Tag protein
We engineered in a FLAG protein tag in the 3’ end of ComD by including the sequence in comD extraction primer.
Forward primer to extract comD to clone into pSB1C3:
TCTGGAGAATTCGCGGCCGCTTCTAGATGGATTTATTTGGATTTGGGACGG
[6’cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comD]

3’ primer to extract comD with engineered FLAG tag gene sequence:
GCCGGACTGCAGCGGCCGCTACTAGTATTATTACTTGTCGTCATCGTCTTTGTAGTCTCATTCAAATTCCCTCTTAAATCTAATGAT
[6’ cap][21’ RFC10 suffix][6’ reverse complement stop codon][25’ reverse complement FLAG protein ][30 reverse complement Streptoccocus pneumoniae/NCTC7465/comD]


comE was extracted from pKHS-come kindly sent to us by ??. Extraction was done using the following primers:
Forward primer to extract comE to clone into pSB1C3:
TCTGGAGAATTCGCGGCCGCTTCTAGATGAAAGTTTTAATTTTAGAAGATG
[6’ cap][20’ RFC10 prefix][25’ Streptoccocus pneumoniae/NCTC7465/comE]

Reverse primer to extract comE to clone into pSB1C3:
GCCGGACTGCAGCGGCCGCTACTAGTATCACTTTTGAGATTTTTTCTCTAA
[6’ cap][21’ RFC10 suffix][24’reverse complement Streptoccocus pneumoniae/NCTC7465/comE]

come contained an illegal SpeI site, so we designed primers for site-directed mutagenesis:
Mutagenesis forward primer: CGCTATTATCGTCTTTATCACTAGCCGATCAGAGTTTGCGACTCTAAC

Mutagenesis reverse primer: GTTAGAGTCGCAAACTCTGATCGGCTAGTGATAAAGACGATAATAGCG

However, site-directed mutagenesis attempts were unsuccessful, so the gene was extracted in two parts using (i) come forward primer & mutagenesis reverse primer; (ii) come reverse primer & mutagenesis forward primer. The two fragments were then combined through Gibson Assembly.