Difference between revisions of "Part:BBa K3431015"

Revision as of 13:46, 26 October 2020

T7 Promoter + RBS + Thermotoga maritima Invertase + T7 Terminator

Introduction

This is a composite part consists of 4 basic parts: T7 promoter (BBa_I719005), RBS (BBa_B0034), invertase (BBa_K3431000), and T7 terminator (BBa_K731721). The T7 promoter (BBa_I719005), RBS (BBa_B0034) and T7 terminator (BBa_K731721) are the essential genetic elements for the in vitro transcription/ translation with PURExpress protein synthesis kit. Invertase, also called β-D-fructofuranosidas, is an important enzyme for the hydrolysis of sucrose to a mixture of glucose and fructose. It is commonly used as reporter proteins since its product, glucose, can be easily detected with a personal glucose meter(PGM) ^[1] , which has a high utilization rate in the public. According to the previous research, Thermotoga maritima Invertase (invertase from Thermotoga maritima) (TmINV) has been proven to have high activity and thermo-stability compared to the commonly used commercial yeast invertase. ^[2]

Characterization of the activity

The 2020 iGEM CSMU_Taiwan has used both models and experiments to measure the activity of invertase. For the modeling, we predicted the kinetics of the invertase with MATLAB. To see the detailed modeling result, please check our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model . As for the wet lab experiments, we produced the invertase with the PURExpress protein synthesis kit. Then we measured its reaction velocity under different sucrose concentrations.The invertase enzymatic reaction was executed at 55℃, which is the best activity temperature for commercial yeast invertase commonly used in PGM-based reaction ^{1 2} . The result of the model and the experiment is shown below.

Reference

1. Xiang, Y., & Lu, Y. (2011). Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nature chemistry, 3(9), 697–703. https://doi.org/10.1038/nchem.1092
2. Du, Y., Hughes, R. A., Bhadra, S., Jiang, Y. S., Ellington, A. D., & Li, B. (2015). A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers. Scientific reports,5, 11039. https://doi.org/10.1038/srep11039

Sequence and Features