sb0c00064_si_001.pdf (245.35 kB)
Toolkit Development for Cyanogenic and Gold Biorecovery Chassis Chromobacterium violaceum
journal contribution
posted on 2020-03-19, 18:42 authored by Lu Ting Liow, Maybelle Kho Go, Matthew Wook Chang, Wen Shan YewChromobacterium
violaceum has been of interest
recently due to its cyanogenic ability and its potential role in environmental
sustainability via the biorecovery of gold from electronic
waste. However, as with many nonmodel bacteria, there are limited
genetic tools to implement the use of this Gram-negative chassis in
synthetic biology. We propose a system that involves assaying spontaneous
antibiotic resistances and using broad host range vectors to develop
episomal vectors for nonmodel Gram-negative bacteria. These developed
vectors can subsequently be used to characterize inducible promoters
for gene expressions and implementing CRISPRi to inhibit endogenous
gene expression for further studies. Here, we developed the first
episomal genetic toolkit for C. violaceum consisting
of two origins of replication, three antibiotic resistance genes,
and four inducible promoter systems. We examined the occurrences of
spontaneous resistances of the bacterium to the chosen selection markers
to prevent incidences of false positives. We also tested broad host
range vectors from four different incompatibility groups and characterized
four inducible promoter systems, which potentially can be applied
in other Gram-negative nonmodel bacteria. CRISPRi was also implemented
to inhibit violacein pigment production in C. violaceum. This systematic toolkit will aid future genetic circuitry building
in this chassis and other nonmodel bacteria for synthetic biology
and biotechnological applications.