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Non-antibiotic selectable and counter-selectable markers:
Plac-RFP-SacB
Background
SacB is levansucrase enzyme from Bacillus subtilis (Gay, Coq, Strinmetz, Ferrari, & Hoch, 1983) which converts sucrose into polymers which are lethal to Esherichia coli (French & Kowal, 2010). This part was deposited in the registry by Team Edinburgh 2010 and can be used as a counter selectable marker (French & Kowal, 2010). Our aim is to improve the part by accessing the counter selection efficiency.
Cloning
The plac-RFP fragment was obtained from standard biobrick plasmid and inserted in front of the SacB biobrick. It was further proceed to add this fragment in front of the SacB biobrick. Method. The construct was confirmed with sequencing.
PCR of PSBIK3 plasmid (Kanamycin resistance) with psBNX3 insF2 forward primer (specific for biobrick prefix) and dsred r2 reverse primer (specific for RFP) was prepared in order to obtain plac-RFP fragment.
Figure 17: DNA gel of the PCR product from psBIK3 amplification with primers specific for the biobrick prefix and RFP. The band is abound 1 kb which corresponds to the expected size of plac-RFP.
Close the plasmid.
The plac-RFP PCR product was purified and digested with EcoRI HQ and SpeI. The SacB biobrick deposited in 2010 (French & Kowal, 2010) was digested with EcoRI and XbaI. These were ligated together after purification. E.coli cells were transformed with the ligation. The red transformants were minipreped, digested with EcoRI HQ in order to linearise them and with EcoRI HQ and PStI in order to check the size of the insert.
Figure 18: DNA gel of miniprepped red clones of plac-RFP-SacB ligation transformants which were linearized. The band is around 4.5 kb which corresponds to psBIC3 (2kb)+ +
Figure 19 The same clones were digested with EcoRI HQ and PstI to check the size of the insert. The band us around 2.5 kb which corresponds to SacB (1.5 kb)+ plac-rfp (1kb).
Close the method.
Sequencing results
Forward primer:
Ctttaaaaaaaatcccttagctttcgctaaggtgatttctggaattcgcggccgcttctagagcaatac
gcaaaccgtttcaccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgga
aagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacact
ttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacatactagataaaga
ggagaaatactagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcttatgga
aggttccgttaactgtcactagttcgaaatcgaaggtgaatgtgaaggtcgtccgtactaaggtaccca
gactgctaaactgaaagttactaaag
Reverse primer:
aggggccttaaacataaacttttcggttttagaaaagggcagggtggtgacaccttgcccttttttgcc
ggactgcagctactagtaatttatttgttaactgttaattgtccttgttcaaggatgctgtctttgaca
acagatgttttcttgcctttgatgttcagcaggaagcttggcgcaaacgttgattgtttgtctgcgtaa
aatcctctgtttgtcatatagcttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgt
gagtaattaaaggttacatcgttaggatcaagatccatttttaacacatggcctgttttgttcagcggc
ttgtatgggccatttaaagaattagaaactttaccaagcatgttaatatcgttagacttatttccgtca
atccttatttttgatccgcgggagtcatttaacaggtaccatttgccgttcattttattttcgttcgcg
cgtctatttctttttgttactttgttttatgcaatcacgttttcattccttttttaattttgtatcatcgt
Close the sequencing results.
Characterization
