Part:BBa_K2090000
phoA->LbCHI31->Flag tag
Group: TPHS_San_Diego 2018
Type of Improvement: Informational
Authors: Joshua Chung, Allison Bien
Summary of Improvement:
The 2016 TP_CC_SanDiego iGEM team used part BBa_K2090000 which was a PhoA-LbChi31-Flag tag construct. Our 2018 IGEM team took this construct and improved upon it by adding a GST sequence to improve efficiency and ease of protein purification. We also used a different strain of chitinase (ChiA of Serratia Marcescens) in hopes of improving the efficiency of the enzyme. We kept the same signal peptide (PhoA) and the same protein tag (flag-tag). The 2016 TP_CC_SanDiego iGEM did a research project to observe the chitinolytic activity of LbChi31 and LbChi32 Chtiinase. Our 2018 TPHS_San_Diego iGEM team did an improvement on this teamś project. The improvements that we made on this project are mostly changing the sequence to optimize the efficiencies to 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_SanDiego 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.
Uploads: (The results are embedded in the upload: SDS Page)
Uploads: 3D model of TPHS_San_Diego 2018 Serratia Marcescens Chitinase Protein
Team: TP_CC_SanDiego 2016
Author: Anthony Khang
Summary of Part:
This linked protein encodes a Limonium bicolor-derived LbCHI32 chitinase enzyme with the signal sequence of phoA (alkaline phosphatase) linked upstream of the chitinase gene by a glycine-serine linker. Downstream from the LbCHI32 gene is a FLAG tag used for protein expression and production analysis via immunoprecipitation.
The translated protein encodes a chitinolytic enzyme that can be secreted extracellularly by Escherichia coli; the presence of this protein can be identified using anti-FLAG antibodies.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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