Composite

Part:BBa_K3431015

Designed by: Jian-An Pan, Cheng-Yang Ma, Yi-Ching Chen, Shen-Lin Chen, Huan-Jui Chang   Group: iGEM20_CSMU_Taiwan   (2020-07-23)
Revision as of 13:46, 26 October 2020 by Jerry252ray (Talk | contribs) (→‎Characterization of the activity)


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.

Figure 1. The initial velocity of the invertase enzymatic reaction under different sucrose concentrations. The green line refers to the regression curve of experimental data, and the blue line refers to the invertase activity model.

Results The initial velocity of the reaction increases as the concentration of the substrate (sucrose) rises. The trend of experimental data fitted our model.

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1356
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1127
    Illegal BamHI site found at 1257
    Illegal XhoI site found at 1328
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 928
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 459


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