Difference between revisions of "Part:BBa K3431015"

 
(→‎Characterization of the activity)
 
(8 intermediate revisions by 3 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K3431015 short</partinfo>
 
<partinfo>BBa_K3431015 short</partinfo>
  
−
This is a protein generator that expresses a hydrolytic protein - Beta-Fructosidase (Invertase, BBa_K3431000). We added T7 Promoter (BBa_I719005) and RBS (BBa_B0034) upstream as well as T7 terminator (BBa_K731721) downstream of Beta-Fructosidase to insure its ability of transcription and translation in PURExpress in vitro Protein Synthesis Kit.  
+
=== 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) <sup>[1]</sup> , which has a high utilization rate in the public. According to the previous research, <i>Thermotoga maritima</i> Invertase (invertase from <i>Thermotoga maritima</i>) (TmINV) has been proven to have high
 +
activity and thermo-stability compared to the commonly used commercial yeast invertase. <sup>[2]</sup>
 +
 
 +
===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 <sup>[1]</sup> <sup>[2]</sup> . The result of the model and the experiment is shown below.
 +
<html>
 +
<br>
 +
<div style="width=100%; display:flex; align-items: center; justify-content: center">
 +
<img src="https://static.igem.org/mediawiki/parts/e/ef/T--CSMU_Taiwan--ES-concentration.jpeg" style="width:70%">
 +
</div>
 +
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.
 +
<br>
 +
</html>
 +
<br>
 +
<b>Results</b>  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., &amp; 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<br>
 +
2. Du, Y., Hughes, R. A., Bhadra, S., Jiang, Y. S., Ellington, A. D., &amp; 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
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:56, 27 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.

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