The 2016 TP-CC iGEM did a research project to observe the chitinolytic activity of LbChi31 and LbChi32 Chitinase. Our 2018 TPHS_San_Diego iGEM team made an improvement on this team's project. The type of improvement that we made was informational: we changed the sequence to optimize the efficiency of our project. We improved the construct by using a sequence from a more efficient strain of chitinase known as Serratia Marcescens chitinase. This strain has been proven to be more effective in previous studies and is effective against a wider range of fungi species. We also added a GST (glutathione S-transferase) sequence in order to allow for protein purification. During our experiment we attempted to produce Serratia Marcescens chitinase on a small scale, the GST sequence was added so that we could use glutathione beads to bind to the protein and thus we could purify the protein via pull down of the beads. This is an improvement upon the 2016 TP-CC iGEM team’s project as the previous team’s sequence did not include a GST (or protein tag for protein purification) sequence and also used a less efficient strain of chitinase. Additionally, our team designed a new 3D model of the crystal structure of the Serratia Marcescens chitinase protein that was included in our new GST-ChiA-FLAG part. The 2016 TP_CC_SanDiego iGEM team did not use a 3D diagram to model their protein, but the 2018 TPHS_San_Diego iGEM created a 3D image of our protein. The 3D model is pictured below. We also used Western Blot to find the optimal concentration of arabinose to maximize induction efficiency which utilizes the AraC operon that is present in both the 2016 and 2018 project to a greater extent.
Rendering of our protein model.