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Applications of BBa_K523002

UMA_MALAGA 2022 Improvement

Group: UMA_MALAGA
Author: Molina Calvo, Alonso

This year we decided to improve BBa_K523002 by deleting the native RBS and optimizing the whole sequence. We used our new part (BBa_K4368002) to have an enzymatic complex named CAZymes with cenA and cex to degrade cellulose into glucose.

Characterization

The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a chloramphenicol resistant plasmid backbone (Cm). Transforming bacteria were created with this plasmid and seeded on LB-Agar+Cm plates. After growth, colonies were selected based on their color (white) and DNA extraction was performed using the Promega PureYield Plasmid Miniprep System kit. The resulting DNA is used for further digestion with EcoRI and PstI. The digests are then run on a 0.75% agarose gel at 90 mV voltage and constant amperage. BioRad brand RedSafe is used as an intercalating developing agent.

Enzyme digestion

Conclusion

We succeded in the selection of transformed bacteria with this gene. We observed degradation of celulose in plates using the red congo assay and measuring the hidrolisis halo.

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