Part:BBa_K1321335:Experience

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Applications of BBa_K1321335

BBa_K1321335 was digested with XbaI and PstI and cloned into a medium-to-low copy number plasmid (pSB3K3) containing the inducible pLAC promoter. The resulting construct, verified by GreenTaq Colony PCR, was subsequently sub-cloned into BBa_K1321336-containing electrocompetent cells. These were plated on LB Agar+Chloramphenicol plates, then incubated overnight at 37 degrees. 50 mL Falcon tubes containing 5 ml LB supplied with 50 ug/ml Chloramphenicol were inoculated with a selection of freshly grown colonies for further restriction analysis and gene sequencing verification.

BBa_K1321336 was characterised in conjunction to an additional pLAC-inducible expression system containing AcsC and AcsD (optimised coding sequences submitted as Part BBa_K1321335) cloned into a medium-to-low copy number plasmid, pSB3K3. The functions of AcsC and AcsD are yet not very well known but are believed to play a crucial role in cellulose crystallisation and secretion into the extracellular space. Cellulose production was assayed by plating transformed cells on Congo Red assay plates containing 20uM CR, 0.5mM IPTG, 0.1% Arabinose, 1% Glucose, 25ug/ml Chloramphenicol and 25ug/ml Kanamycin. Cellulose-producing E.coli colonies turned red in the presence of CR.

Figure 1 - Congo Red assay, positive colonies

Figure 2 - Congo Red assay, negative control

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