Difference between revisions of "Part:BBa K3141001"
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<partinfo>BBa_K3141001 short</partinfo>
 
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HPI gene encodes the synthesis of proinsulin.It contains three exons which encodes A chain, B chain and C peptide. Based on HPI gene, we optimized its codon for higher expression in B.Subtilis. What’s more, we alternated a short region on HPI gene to increase its protein synthesis and secretion.
 
HPI gene encodes the synthesis of proinsulin.It contains three exons which encodes A chain, B chain and C peptide. Based on HPI gene, we optimized its codon for higher expression in B.Subtilis. What’s more, we alternated a short region on HPI gene to increase its protein synthesis and secretion.
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=== Use of Biology ===
 
=== Use of Biology ===
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Proinsulin is a precursor to insulin biosynthesis which can bind to insulin receptor in the liver membrane, to stimulate livers to convert glucose to glycogen. Injections of proinsulin intravenously in animal or man results in a decrease in blood glucose. This means that proinsulin has similar functions of lowering blood glucose level as insulin. In human body, proinsulin is synthesized in pancreatic βcells to enable the downstream insulin secretion into blood. As the only blood glucose reducer, insulin operates mainly through the regulation of hepatic metabolism for the glucose uptake by lever cells[ Paul M. Titchenell, et al:Unraveling the regulation of Hepatic Metabolism by Insulin. Trends Endocrinol Metab. 2017 Jul;28(7):497-505  ]. When the level of glycogen reduces and the concentration of insulin rises, liver cells start to increase the uptake of glucose, synthesis of glycogen, suppression of hepatic glucose production(HGP), and the induction of the synthesis of fatty acids for energy storage and further utilization[ LinHV, et al.:Hormonal Regulation of Hepatic glucose Production in Health and Disease. Cell Metab.2011;14:9-19].
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Proinsulin is a precursor to insulin biosynthesis which can stimulate livers to convert glucose to glycogen through binding to insulin receptor in the liver membrane.In human body, proinsulin is synthesized in pancreatic βcells to enable the downstream insulin secretion into blood. When the blood glucose concentration increases, insulin synthesis accelerates. At the same time, a small portion of proinsulin without protease hydrolysis enters the blood circulation. As the only blood glucose reducer, insulin operates mainly through the regulation of hepatic metabolism for the glucose uptake by lever cells. When the level of glycogen reduces and the concentration of insulin rises, liver cells start to increase the uptake of glucose, synthesis of glycogen, suppression of hepatic glucose production(HGP), and the induction of the synthesis of fatty acids for energy storage and further utilization. We can assume proinsulin to have similar functions but is one tenth as affective as insulin.
  
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=== Characterization ===
 
 
To test the expression efficiency, this gene is transformed into pHY-P43 and is expressioned in ''B.subtilis''.
 
To test the expression efficiency, this gene is transformed into pHY-P43 and is expressioned in ''B.subtilis''.
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Experimental method:
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#Construction of bacteria:Original HPI gene and modified HPI gene are both transformed into bacteria B.subtilis
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#Fermentation
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#protein extraction(through mechanical attrition)
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#Protein Gel Electrophoresis
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==== Experiment Result ====
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<u>Protein electrophoresis:</u>
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<p>Figure 1 and Figure 2 show the result of protein electrophoresis to both unmodified bacteria and the bacteria that contained recombinant DNA.</p>
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<html>
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<center>
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<figure>
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<img width="70%" src="https://2019.igem.org/wiki/images/7/7b/T--BSC_United--empty.png">
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</figure>
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<h5>Figure 1: the SDS-PAGE result of bacteria with unmodified HPI gene(BBa_K1328003).Trial 1 is negative control--the supernatant remained after the collection of WB800N. Trial 2-3 are supernatant of sample 1 and 2. Trial 4-6 are 10-times-diluted supertanants of trial 1-3.</h5>
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</center>
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</html>
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<p>Through the figure 1 it is known that bacteria with original HPI gene secreted a small amount of proinsulin that is not enough to be perceived by naked eyes.</p>
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<html>
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<center>
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<figure>
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<img width="70%" src="https://2019.igem.org/wiki/images/c/ca/T--BSC_United--notempty.png">
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</figure>
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<h5>Figure 2: SDS-PAGE result of bacteria with codon-optimized and N-terminal-modified HPI gene(BBa_K3141001).Trial 1 is negative control--the supernatant remained after the collection of WB800N. Trial 2-3 are supernatant of sample 13 and 16. Trial 4-6 are 5-times-diluted supertanants of trial 1-3. </h5>
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</center>
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</html>
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<p>The target protein bars existed in expected size, where proinsulin is about to shown--at 10.5kD to 14kD. Bacteria with modified HPI gene produced large amount of target protein(as shown in figure 2). As a result,it is found that the modification to HPI sequence increased the production of protein.</p>
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K3141001 SequenceAndFeatures</partinfo>
 
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3141001 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3141001 parameters</partinfo>
 
<partinfo>BBa_K3141001 parameters</partinfo>
 
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Latest revision as of 22:36, 20 October 2019

Proinsulin secretion; Highly expressed

HPI gene encodes the synthesis of proinsulin.It contains three exons which encodes A chain, B chain and C peptide. Based on HPI gene, we optimized its codon for higher expression in B.Subtilis. What’s more, we alternated a short region on HPI gene to increase its protein synthesis and secretion.

Use of Biology

Proinsulin is a precursor to insulin biosynthesis which can stimulate livers to convert glucose to glycogen through binding to insulin receptor in the liver membrane.In human body, proinsulin is synthesized in pancreatic βcells to enable the downstream insulin secretion into blood. When the blood glucose concentration increases, insulin synthesis accelerates. At the same time, a small portion of proinsulin without protease hydrolysis enters the blood circulation. As the only blood glucose reducer, insulin operates mainly through the regulation of hepatic metabolism for the glucose uptake by lever cells. When the level of glycogen reduces and the concentration of insulin rises, liver cells start to increase the uptake of glucose, synthesis of glycogen, suppression of hepatic glucose production(HGP), and the induction of the synthesis of fatty acids for energy storage and further utilization. We can assume proinsulin to have similar functions but is one tenth as affective as insulin.

To test the expression efficiency, this gene is transformed into pHY-P43 and is expressioned in B.subtilis. Experimental method:

  1. Construction of bacteria:Original HPI gene and modified HPI gene are both transformed into bacteria B.subtilis
  2. Fermentation
  3. protein extraction(through mechanical attrition)
  4. Protein Gel Electrophoresis

Experiment Result

Protein electrophoresis:

Figure 1 and Figure 2 show the result of protein electrophoresis to both unmodified bacteria and the bacteria that contained recombinant DNA.

Figure 1: the SDS-PAGE result of bacteria with unmodified HPI gene(BBa_K1328003).Trial 1 is negative control--the supernatant remained after the collection of WB800N. Trial 2-3 are supernatant of sample 1 and 2. Trial 4-6 are 10-times-diluted supertanants of trial 1-3.

Through the figure 1 it is known that bacteria with original HPI gene secreted a small amount of proinsulin that is not enough to be perceived by naked eyes.

Figure 2: SDS-PAGE result of bacteria with codon-optimized and N-terminal-modified HPI gene(BBa_K3141001).Trial 1 is negative control--the supernatant remained after the collection of WB800N. Trial 2-3 are supernatant of sample 13 and 16. Trial 4-6 are 5-times-diluted supertanants of trial 1-3.

The target protein bars existed in expected size, where proinsulin is about to shown--at 10.5kD to 14kD. Bacteria with modified HPI gene produced large amount of target protein(as shown in figure 2). As a result,it is found that the modification to HPI sequence increased the production of protein.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 198
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters

<