Due to the need to find a solution to a local problem such as contamination by vegetal wastes, a gene
construct
was designed as an alternative to transform these products into a value-added substance, starch. For this
purpose, we used optimized synthetic gene constructs from Escherichia coli and Cellulomonas fimi, which were
subsequently transformed into E.coli bacteria.
Once the final construct based on two plasmids, pSBC1Cel1 and pSBA1Sta1, was obtained, a search was carried
out
to determine the existing detection methods that would allow verification of this construct. Through a
literature search, it was possible to determine that simple tests such as Red Congo Assay, as well as
staining
with lugol and glucose oxidase test (GOD) allowed verifying the correct functioning of the composition
created.
They were accompanied by a final fluorescence revealed due to emission at the wavelengths corresponding to
RFP
and GFP.
In silico design
For this purpose, the different gene constructs that make up our glucose-sensing-based regulatory mechanism
(Parts) were designed computationally in a software named Benchling.
Firstly, a sequence of an extracellular export motor protein was introduced into the cenA and cex constructs
that in conjunction with the bglX gene allows extracellular cellulose to be degraded into glucose.
Secondly, codon optimization was performed on the gene encoding glgC16 by removing an EcoRI restriction
target. In addition, the basic part of bglX (BBa_K523002) has been modified by removing an overlapping
native RBS.
Establishment of a protocol for the final construct
Our construct is based on the existence of only two plasmids, pSBC1Cel1 and pSBA1Sta1, which combine all the
genes required for starch production. For this purpose, a paired ligation protocol based on the BioBrick®
Assembly Kit has been designed. For more information on how this procedure or every other one of our
experimentation was performed, please click on the button below to get a copy of our protocols.
Screening tests
For final testing that our verified construct is working optimally, colorimetric (congo red and lugol) and
enzymatic (glucose oxidase) assays are performed. If you require more information on how these procedures
have been approached, please visit the Verification section of Experiments.
