Difference between revisions of "Part:BBa K528004"

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__NOTOC__
 
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<partinfo>BBa_K528004 short</partinfo>
 
<partinfo>BBa_K528004 short</partinfo>
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<h2>Improvement</h2>
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This part is improved to Part:[https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139017 BBa_K3139017]
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2019 NAU-CHINA obtained the sequence of this part and requested the plasmid from them.We planned to insert an A/U rich region in the upstream of the RBS of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K528004 BBa_K528004], and measure the florescence intensity of mRFP1. If the florescence intensity rises as the region inserted, the improvement shall be proved to be feasible.
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===The mechanism of A/U-rich 5'-UTR to promote protein synthesis===
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<html>
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<img src="https://2019.igem.org/wiki/images/5/5c/T--NAU-CHINA--UTR1.png"width="600"/>
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</html><BR>
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Fig.1. The mechanism of A/U-rich 5'-UTR to promote protein synthesis.<BR>
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(1) The A/U-rich 5'-UTR induces the ribosomal protein S1 to bind to the RBS, promoting the translation process and accelerating the protein synthesis.<BR>
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(2) Induced ribosomes bind to the mRNA more frequently which stabilizes the mRNA, making the mRNA to be functional in a longer period, promoting the protein synthesis.<BR>
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===UTR works well in E. coli===
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<html>
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<img src="https://2019.igem.org/wiki/images/7/7a/T--NAU-CHINA--UTR2.png"width="850"/>
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</html><BR>
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Fig.2. Data obtained by plate reader.<BR>
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(A)Measurement of OD600nm of DH5α.<BR>
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(B)Measurement of RFP fluorescence intensity at 611nm (excitation at 584nm) of DH5α.<BR>
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The figure shows that the distinction among OD600nm data were not clear between 2 treatments, which means that we can still compare the fluorescence intensity as they probably were in the same growth phase.<BR>
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The sample with UTR sequence increases the secretion to approximately 2-fold, which means RFP characterize level has significant improvement. It shows that UTR greatly improves the efficiency of protein characterization.<BR>
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In conclusion, the mRFP1 is synthesized more rapidly. As the part BBa_K528004 is a measurement device of RBS [measuring strength by measuring florescence intensity, then setting a standard values such as the florescence intensity of B0034, comparing the data and calculating the (florescence intensity sample) / (florescence intensity standard)], the measurement process could be much faster as the protein synthesis promoted. Besides, strength gaps among different RBS will also rise as the mRFP1 synthesized faster.
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===Reference===
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[1] Khosa S , Scholz R , Schwarz C , et al. An A/U-rich enhancer region is required for high-level protein secretion through the HlyA Type I secretion system[J]. Applied and Environmental Microbiology, 2017:AEM.01163-17. Sequence and Features
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<h2></h2>
  
 
This device was used to determine BBa_B0032 strength relative to BBa_B0034.
 
This device was used to determine BBa_B0032 strength relative to BBa_B0034.
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===Measurements===
 
===Measurements===
  
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Using this construct we have determined that strength of BBa_B0031 with RFP is 65,56% relative to BBa_B0034 with RFP.
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Using this construct we have determined that strength of BBa_B0032 with RFP is 65,56% relative to BBa_B0034 with RFP.
  
 
All our RFP measurement results are summarized on the chart below:
 
All our RFP measurement results are summarized on the chart below:

Latest revision as of 01:26, 22 October 2019

RBS measurement device J23100 B0032 RFP

Improvement

This part is improved to Part:BBa_K3139017

2019 NAU-CHINA obtained the sequence of this part and requested the plasmid from them.We planned to insert an A/U rich region in the upstream of the RBS of BBa_K528004, and measure the florescence intensity of mRFP1. If the florescence intensity rises as the region inserted, the improvement shall be proved to be feasible.

The mechanism of A/U-rich 5'-UTR to promote protein synthesis


Fig.1. The mechanism of A/U-rich 5'-UTR to promote protein synthesis.
(1) The A/U-rich 5'-UTR induces the ribosomal protein S1 to bind to the RBS, promoting the translation process and accelerating the protein synthesis.
(2) Induced ribosomes bind to the mRNA more frequently which stabilizes the mRNA, making the mRNA to be functional in a longer period, promoting the protein synthesis.

UTR works well in E. coli


Fig.2. Data obtained by plate reader.
(A)Measurement of OD600nm of DH5α.
(B)Measurement of RFP fluorescence intensity at 611nm (excitation at 584nm) of DH5α.
The figure shows that the distinction among OD600nm data were not clear between 2 treatments, which means that we can still compare the fluorescence intensity as they probably were in the same growth phase.
The sample with UTR sequence increases the secretion to approximately 2-fold, which means RFP characterize level has significant improvement. It shows that UTR greatly improves the efficiency of protein characterization.

In conclusion, the mRFP1 is synthesized more rapidly. As the part BBa_K528004 is a measurement device of RBS [measuring strength by measuring florescence intensity, then setting a standard values such as the florescence intensity of B0034, comparing the data and calculating the (florescence intensity sample) / (florescence intensity standard)], the measurement process could be much faster as the protein synthesis promoted. Besides, strength gaps among different RBS will also rise as the mRFP1 synthesized faster.

Reference

[1] Khosa S , Scholz R , Schwarz C , et al. An A/U-rich enhancer region is required for high-level protein secretion through the HlyA Type I secretion system[J]. Applied and Environmental Microbiology, 2017:AEM.01163-17. Sequence and Features


This device was used to determine BBa_B0032 strength relative to BBa_B0034.

Construct design

The device consists of BBa_K299502 expression vector J23100+B0032. E1010 RFP was used as reporter protein.

Authors

Clonning:

  • Paweł Urbański
  • Elżbieta Jankowska
  • Krzysztof Szczepaniak
  • Dorota Kaja Sabat

The lab work was supervised by:

  • Michał Lower
  • Ania Olchowik

Measurements

Using this construct we have determined that strength of BBa_B0032 with RFP is 65,56% relative to BBa_B0034 with RFP.

All our RFP measurement results are summarized on the chart below:

Wyk6.png

For more info please look at Team Warsaw 2011 [http://2011.igem.org/Team:Warsaw/RBSmeasurement].

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 617
    Illegal AgeI site found at 729
  • 1000
    COMPATIBLE WITH RFC[1000]