Discipline: Technology and Engineering
Subcategory: Chemical/Bimolecular/Process Engineering
Room: Exhibit Hall A
Agata Turula - City College of New York
Co-Author(s): Veronica Montgomery, Georgia Institute of Technology, Atlanta, GA; Mark R. Prausnitz, Georgia Institute of Technology, Atlanta, GA
Chronic wounds affect 6.5 million people in the United States and the cost of treatment amounts to $25 billion annually. They are caused by long term pressure on an area of skin and are known to be very painful. Collagenase Santyl ointment is an important aspect of chronic wound healing since it debrides necrotic tissue. Collagenase is an enzyme derived from Clostridia histolyticum that breaks down denatured collagen. However, clostridia cannot produce the large amounts of collagenase needed for medical use and they are generally regarded as dangerous due to their pathogenic origin. Bacillus subtilis and Escherichia coli are safer bacteria to work with and yield higher quantities, so they are often genetically engineered to produce collagenase. B. subtilis is the desired bacteria for the secretion of collagenase because it is gram positive bacteria and delivers proteins through its one lipid bilayer more effectively than gram negative bacteria, which has two. Attempts to engineer B. subtilis to secrete collagenase have been done in the past, but plasmid stability was not maintained. For this project, a plasmid that showed good stability in previous research was used to create recombinant DNA containing collagenase and a secretion tag. The specific gene ColH was inserted into the plasmid and the secretion tag used was PenP. These DNA segments were first amplified through polymerase chain reactions (PCR), and put together through Gibson assembly. Then, various tests where done to assess if the plasmid was correctly assembled. The plasmid was transformed into E. coli and plated on an antibiotic. This checked if the bacterial cells received the plasmid, since the plasmid has an antibiotic resistance gene in its backbone. Next, colony PCR was performed on the transformed cells to check the length of a known section of the plasmid. Restriction digest cuts the plasmid at two specific sites to further confirm the correct length of the inserted segments. . Lastly, the plasmid was sequenced to verify the order of the nucleic acids expected. Future work will include transforming the plasmid into Bacillus subtilis and doing a colorimetric secretion assay to determine if the bacteria are secreting active collagenase. Additional steps may include screening different secretion tags to test which one results in successful secretion of collagenase.
Funder Acknowledgement(s): Thank you to NSF and Intel for financial support, as well as the Prausnitz Lab for Drug Delivery, Styczynski Lab, and the SURE Program at Georgia Tech.
Faculty Advisor: Mark Prausnitz, email@example.com
Role: I created a plasmid containing a backbone, secretion tag, and collagenase gene. I did this through polymerase chain reactions (PCR) and Gibson assembly. I then assessed my plasmid by transforming it, conducting colony PCR and miniprep, and then restriction digest. I also prepared the sample to be sent for sequencing. The process of constructing the plasmid involved troubleshooting PCR to increase the concentration of the backbone in my samples so that bacterial cells could receive the plasmid.