Difference between revisions of "Part:BBa K3863008"

 
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<partinfo>BBa_K3863008 short</partinfo>
 
<partinfo>BBa_K3863008 short</partinfo>
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This is a composite part designed to produce bioplastic PHA. 
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PHA synthase 1 from Pseudomonas resinovorans. The protein sequence in this part is based on the K2042001 sequence Evry 2016.
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propionate CoA transferase from Clostridum propionicum.  The protein sequence in this part is based on the K1211001 sequence Yale 2013.
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Phasin attaches to the polyhydroxybutyrate (PHB) granule surface regulating the number and size of PHB granules within a cell. It probably also acts as a regulator affecting the biosynthetic activity of PHB synthase in vivo.
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<!-- Add more about the biology of this part here
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===Usage and Biology===
  
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1. Phac (BBa_K2042001 from iGEM 2016 iGEM16_Evry)
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3863008 SequenceAndFeatures</partinfo>
  
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  1. Function: This is the coding sequence for class II poly(R)-hydroxyalkanoic acid (PHA) synthase 1 from Pseudomonas resinovorans. The enzyme takes the monomer (D)-lactyl-CoA and produce the PLA homopolymer.
 
  
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  2. Full name: Pseudomonas resinovorans PHA synthase 1
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<h2>Validation of construct expression of K3863008</h2>
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2. PCT (BBa_K1211001 from iGEM 2013 iGEM13_Yale)
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https://2021.igem.org/wiki/images/5/58/T--PuiChing_Macau--resultt1.jpg
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<p>Fig 1. Real-time qPCR fold change of reference gene</p>
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<p>To future confirm that our constructs have the expression level, we have performed RT-qPCR
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The RT-qPCR results show that we have successfully assembled target genes K3863008 to the vector (pETDuet).
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</p>
  
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  1. Function: used this enzyme as the first enzyme in the pathway to create PLA or Polylactic acid. However, this biobrick produces (D)-Lactyl-CoA, which is the precursor to all PHAs
 
  
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  2. Full name: Clostridum propionicum propionate CoA transferase
 
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3. Phasin
 
  
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      a. Function:  promote PHA production and Increase PhaC production rate (4 times the original speed)
 
  
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A phasin, it attaches to the polyhydroxybutyrate (PHB) granule surface regulating the number and size of PHB granules within a cell. It probably also acts as a regulator affecting the biosynthetic activity of PHB synthase in vivo.
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<h2>Validation of Bioplastic Production</h2>
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<p>In order to observe the conversion performance, infrared spectroscopic analysis was applied since PHA , PLA and PhaC have their particular functional group in chemical structure, and are performed in particular wavelengths. The absorption peak of PLA is at 1081, 1188, 1364, 1452 , 1751 cm-1[1],  whereas the absorption peak 979, 1057, 1100, 1282, 1723, 2934, 2977cm-1[2]. We have compared and analyse the peak of the wavelength to confirm the bioplastic product we have produced.
 +
In Figure 2, no absorption peak of PHA and PLA was identified, which indicated that the vector (pETDuet Vector) cannot give the desired product. On the other hand, in Figure (3b) and (3c),  the absorption peaks of PHA and PhaC were identified after incubation, which suggested that the BBa_K3863004 (in Figure (3b)) and BBa_K3863008 (in Figure (3c)), the bioplastic performs significantly in the transformation process. Furthermore, the absorption peaks of PHA in Figure (d) were found, which  showed that BBa_K3863007 performs well and was the product that we expected to have. These results have proven that the bioplastic that we have produced can be formed significantly with BBa_K3863004, BBa_K3863008 and BBa_K3863007.
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</p>
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https://2021.igem.org/wiki/images/7/75/T--PuiChing_Macau--result13.jpg
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<p>Figure 2a. IR spectrum of  pETDuet vector</p>
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https://2021.igem.org/wiki/images/1/18/T--PuiChing_Macau--result15.jpg
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<p>Figure 2b. IR spectrum of  BBa_K3863008 </p>
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
−
<!-- -->
 
−
<span class='h3bb'>Sequence and Features</span>
 
−
<partinfo>BBa_K3863008 SequenceAndFeatures</partinfo>
 
  
 +
<h2>Quantitative Measure of Bioplastic Production</h2>
 +
<p>In order to measure the amount of bioplastic produced, we used 20 microgram Nile Red (sigma aldrich N3013) per milliliter of agar to make a Nile agar plate and streak the cell culture (PHA, PhaC, Phasin) on the plate. After incubating for 2 days at 37 celsius in darkness. Stereomicroscope (Nikon SMZ18) is used to measure the intensity of Nile red. </p>
 +
https://2021.igem.org/wiki/images/c/c5/T--PuiChing_Macau--result0000.jpg
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<p>Mean intensity of Nile red for quantitative measure of bioplastic production of (BBa_K3863007(PHA), BBa_K3863004(PhaC), BBa_K3863008(Phasin))</p>
  
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<h2>Reference</h2>
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[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3450024/pdf/12088_2009_Article_31.pdf
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[2]https://www.researchgate.net/figure/Fourier-transform-infrared-FTIR-spectra-of-PLA-PEG-PLA-PEG-blend-and-PLA-PEG-xGnP_fig1_277675367
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3863008 parameters</partinfo>
 
<partinfo>BBa_K3863008 parameters</partinfo>
 
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Revision as of 16:47, 20 October 2021


Phac-flag-PCT(optimized)-HA-phasin-his This is a composite part designed to produce bioplastic PHA. PHA synthase 1 from Pseudomonas resinovorans. The protein sequence in this part is based on the K2042001 sequence Evry 2016. propionate CoA transferase from Clostridum propionicum. The protein sequence in this part is based on the K1211001 sequence Yale 2013. Phasin attaches to the polyhydroxybutyrate (PHB) granule surface regulating the number and size of PHB granules within a cell. It probably also acts as a regulator affecting the biosynthetic activity of PHB synthase in vivo. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1708
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 87
    Illegal BamHI site found at 1506
    Illegal BamHI site found at 3464
    Illegal XhoI site found at 1030
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1060
    Illegal NgoMIV site found at 2125
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 251


Validation of construct expression of K3863008

T--PuiChing_Macau--resultt1.jpg

Fig 1. Real-time qPCR fold change of reference gene

To future confirm that our constructs have the expression level, we have performed RT-qPCR The RT-qPCR results show that we have successfully assembled target genes K3863008 to the vector (pETDuet).



Validation of Bioplastic Production

In order to observe the conversion performance, infrared spectroscopic analysis was applied since PHA , PLA and PhaC have their particular functional group in chemical structure, and are performed in particular wavelengths. The absorption peak of PLA is at 1081, 1188, 1364, 1452 , 1751 cm-1[1], whereas the absorption peak 979, 1057, 1100, 1282, 1723, 2934, 2977cm-1[2]. We have compared and analyse the peak of the wavelength to confirm the bioplastic product we have produced. In Figure 2, no absorption peak of PHA and PLA was identified, which indicated that the vector (pETDuet Vector) cannot give the desired product. On the other hand, in Figure (3b) and (3c), the absorption peaks of PHA and PhaC were identified after incubation, which suggested that the BBa_K3863004 (in Figure (3b)) and BBa_K3863008 (in Figure (3c)), the bioplastic performs significantly in the transformation process. Furthermore, the absorption peaks of PHA in Figure (d) were found, which showed that BBa_K3863007 performs well and was the product that we expected to have. These results have proven that the bioplastic that we have produced can be formed significantly with BBa_K3863004, BBa_K3863008 and BBa_K3863007.

T--PuiChing_Macau--result13.jpg

Figure 2a. IR spectrum of pETDuet vector

T--PuiChing_Macau--result15.jpg

Figure 2b. IR spectrum of BBa_K3863008



Quantitative Measure of Bioplastic Production

In order to measure the amount of bioplastic produced, we used 20 microgram Nile Red (sigma aldrich N3013) per milliliter of agar to make a Nile agar plate and streak the cell culture (PHA, PhaC, Phasin) on the plate. After incubating for 2 days at 37 celsius in darkness. Stereomicroscope (Nikon SMZ18) is used to measure the intensity of Nile red.

T--PuiChing_Macau--result0000.jpg

Mean intensity of Nile red for quantitative measure of bioplastic production of (BBa_K3863007(PHA), BBa_K3863004(PhaC), BBa_K3863008(Phasin))

Reference

[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3450024/pdf/12088_2009_Article_31.pdf [2]https://www.researchgate.net/figure/Fourier-transform-infrared-FTIR-spectra-of-PLA-PEG-PLA-PEG-blend-and-PLA-PEG-xGnP_fig1_277675367