Difference between revisions of "Part:BBa K2982000"

 
 
(11 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2982000 short</partinfo>
 
<partinfo>BBa_K2982000 short</partinfo>
  
−
A coding sequence for wild type PETase from Ideonella sakainesis which is in BioBrick RFC[10] assembly standard and also contains two site for restriction enzymes, XhoI and BamHI to act on. It is codon optimized for Escherichia coli. Can serve as a standard to compare PETase mutants.
+
A coding sequence for wild type PETase from Ideonella sakainesis. It is codon optimized for Escherichia coli. Can serve as a standard for other experiment related to PETase.
  
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
 +
<h1> BBa_K2982000 (Wild Type) </h1>
 +
<p>A coding sequence of the PETase. </p>
 +
<p>This sequence is codon optimized for Escherichia coli, obtained from previous studies done on PETase.[1] It can serve as a standard for other experiment related to PETase, such as activity measurement.
 +
</p>
 +
<h1>Origin and biology</h1>
 +
<p>The enzyme is a hydrolase which degrades polyethylene terephthalate into simple molecules: MHET, BHET, and TPA by cleavage of the ester bond within the polymer. It was originally found in the bacteria Ideonella sakaiensis, which uses PET as a carbon source, and integrates the degradation products into its metabolic cycle.</p>
 +
<h1>Characterisation</h1>
 +
<p>In our experiments, to insert this gene into cells, the PET-21b vector is used due to its high copy number and the presence of T7 promoter and a lac operon. We use DH5ɑ as host cells due to its high insert stability. Then, extracted DNA is transformed into C41(DE3) cells, which we use to perform the protein induction due to the toxic nature of PETase.</p>
 +
<p>After the protein is induced using 0.5mM IPTG, it can be purified and extracted using a column with nickel resin due to a 6X His-Tag fused with PETase outside the globular structure. After purification, SDS-PAGE can be performed to confirm successful expression.</p>
 +
https://2019.igem.org/wiki/images/5/5c/T--HK_GTC--43.jpg
 +
<p> <center> Figure 1: SDS-PAGE of purified Wild Type PETase. A band of around 30 kDa is clearly shown.
 +
</center> </p>
 +
<p>As shown above, the thick band around 30 kDa shows successful expression of PETase.</p>
 +
<p>After protein purification, an enzyme assay was performed to confirm the protein activity. The substrate used is p-nitrophenyl dodecanoate, as the ester bond is similar to that in PETase. The product, p-nitrophenol has a yellow colour. Therefore, activity can be confirmed by measuring optical density at 415nm.</p>
 +
https://2019.igem.org/wiki/images/d/d7/T--HK_GTC--41.jpg
 +
<p>Figure 2: Optical density at 415nm of reaction mixture with Wild Type PETase. A large increase is clearly seen.</p>
 +
https://2019.igem.org/wiki/images/f/fe/T--HK_GTC--42.jpg
 +
<p>Figure 3: Percentage increase of optical density at 415nm for reaction mixture with Wild Type PETase. </p>
 +
<p>As shown, there is a clear increase in optical density, confirming enzyme activity.</p>
 +
<p>[1]:Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., .
 +
. . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic
 +
polyesterase. Proceedings of the National Academy of Sciences, 115(19).
 +
doi:10.1073/pnas.1718804115
 +
</p>
 +
 +
<!-- Add more about the biology of this part here-->
 +
===Usage and Biology===
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 14:38, 21 October 2019


Coding sequence for wild type IsPETase

A coding sequence for wild type PETase from Ideonella sakainesis. It is codon optimized for Escherichia coli. Can serve as a standard for other experiment related to PETase.


BBa_K2982000 (Wild Type)

A coding sequence of the PETase.

This sequence is codon optimized for Escherichia coli, obtained from previous studies done on PETase.[1] It can serve as a standard for other experiment related to PETase, such as activity measurement.

Origin and biology

The enzyme is a hydrolase which degrades polyethylene terephthalate into simple molecules: MHET, BHET, and TPA by cleavage of the ester bond within the polymer. It was originally found in the bacteria Ideonella sakaiensis, which uses PET as a carbon source, and integrates the degradation products into its metabolic cycle.

Characterisation

In our experiments, to insert this gene into cells, the PET-21b vector is used due to its high copy number and the presence of T7 promoter and a lac operon. We use DH5ɑ as host cells due to its high insert stability. Then, extracted DNA is transformed into C41(DE3) cells, which we use to perform the protein induction due to the toxic nature of PETase.

After the protein is induced using 0.5mM IPTG, it can be purified and extracted using a column with nickel resin due to a 6X His-Tag fused with PETase outside the globular structure. After purification, SDS-PAGE can be performed to confirm successful expression.

T--HK_GTC--43.jpg

Figure 1: SDS-PAGE of purified Wild Type PETase. A band of around 30 kDa is clearly shown.

As shown above, the thick band around 30 kDa shows successful expression of PETase.

After protein purification, an enzyme assay was performed to confirm the protein activity. The substrate used is p-nitrophenyl dodecanoate, as the ester bond is similar to that in PETase. The product, p-nitrophenol has a yellow colour. Therefore, activity can be confirmed by measuring optical density at 415nm.

T--HK_GTC--41.jpg

Figure 2: Optical density at 415nm of reaction mixture with Wild Type PETase. A large increase is clearly seen.

T--HK_GTC--42.jpg

Figure 3: Percentage increase of optical density at 415nm for reaction mixture with Wild Type PETase.

As shown, there is a clear increase in optical density, confirming enzyme activity.

[1]:Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115

Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 348
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 304
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 348
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 348
    Illegal AgeI site found at 627
  • 1000
    COMPATIBLE WITH RFC[1000]