Engineered Microbial Consortium for the Efficient Conversion of Biomass to Biofuels Page: 53
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for expression efficiency. The annealing temperature used was 65 C and extension time was set at
20 seconds. The amplicon was verified on DNA gel electrophoresis for the correct size and
thereafter sequenced for correctness.
3.2.4 Amplification of the Alkaline Phosphatase Gene (phoZ) of Streptococcus pyogenes
The alkaline phosphatase gene phoZ from the plasmid pMNN (kindly provided by
Professor Melody Neely) is used in determining the successful expression of genes on the cell
surface. phoZ gene is only active outside the cell and can be measured colorimetrically in the
presence of bromo-4-chloro-3-indolyl phosphate [188]. The gene was amplified using synthetic
primers at an annealing temperature of 55 C and an extension time of 25 seconds. The amplicon
was verified on DNA gel electrophoresis for the correct size and thereafter sequenced verified for
correctness by sequencing.
3.2.5 Cloning and Expression of the Amplicons
Attempts were made to successfully fuse the individual inserts but with no success and so
this aspect was shelved for future investigation. The efficacy of this construct for the consortium
would have been assessed by the cultivation of Z. mobilis in co-culture with C. cellulolyticum on
microcrystalline cellulose relative to a wild-type control [189].53
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Anieto, Ugochukwu Obiakornobi. Engineered Microbial Consortium for the Efficient Conversion of Biomass to Biofuels, dissertation, August 2014; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc699973/m1/64/: accessed July 17, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .