Difference between revisions of "Part:BBa K3863007"

 
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<partinfo>BBa_K3863007 short</partinfo>
 
<partinfo>BBa_K3863007 short</partinfo>
  
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1. PHA Synthase (BBa_K1211002 from iGEM 2013 iGEM13_Yale)
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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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  1. Function: This is the coding sequence for a Polyhydroxyalkanoate synthase from Pseudomonas resinovorans. The enzyme takes the monomer (D)-lactyl-CoA and creates the PLA homopolymer. This sequence includes four mutations which were found to increase yields of PLA (Yang et al.)
 
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  2. Full name: Pseudomonas resinovorans Polyhydroxyalkanoate synthase
 
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2. PCTcp (BBa_K1211001 from iGEM 2016 iGEM16_Evry)
 
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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.This sequence is codon optimized for Pseudomonas putida and includes four amino acid mutations (E130D/S325T/S477G/Q481K) which were found to increase yields of PLA [1,2]
 
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  2. Full name: Pseudomonas resinovorans PHA synthase 1
 
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  lac operator  RBS  PHA Synthase (BBa_K1211002 )  Flag tag  T7 promotor  lac operator  RBS  PCTcp  HA tag  T7 terminator f1 ori Ampr      Ampr promotor  ori rop    lacI    lacI promotor  T7 promotor
 
  
 
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<partinfo>BBa_K3863007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3863007 SequenceAndFeatures</partinfo>
  
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<h2>Validation of construct expression of K3863007</h2>
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https://2021.igem.org/wiki/images/d/da/T--Puiching_Macau--PHA.png
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<p>1a) PHA (BBa_K3863007) qPCR</p>
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https://2021.igem.org/wiki/images/3/36/T--PuiChing_Macau--PCT.png
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<p>1b) PCT (BBa_K3863007) qPCR</p>
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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).</p>
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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/e/ed/T--PuiChing_Macau--result14.jpg
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<p>Figure 2b. IR spectrum of  BBa_K3863004 </p>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 16:37, 20 October 2021


PHA-flag-PCT-HA

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.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1411
    Illegal PstI site found at 25
    Illegal PstI site found at 118
    Illegal PstI site found at 208
    Illegal PstI site found at 280
    Illegal PstI site found at 742
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1411
    Illegal PstI site found at 25
    Illegal PstI site found at 118
    Illegal PstI site found at 208
    Illegal PstI site found at 280
    Illegal PstI site found at 742
    Illegal NotI site found at 1708
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1411
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1411
    Illegal PstI site found at 25
    Illegal PstI site found at 118
    Illegal PstI site found at 208
    Illegal PstI site found at 280
    Illegal PstI site found at 742
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1411
    Illegal PstI site found at 25
    Illegal PstI site found at 118
    Illegal PstI site found at 208
    Illegal PstI site found at 280
    Illegal PstI site found at 742
    Illegal NgoMIV site found at 1167
    Illegal NgoMIV site found at 1212
    Illegal AgeI site found at 448
    Illegal AgeI site found at 853
    Illegal AgeI site found at 1486
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 251

Validation of construct expression of K3863007

T--Puiching_Macau--PHA.png

1a) PHA (BBa_K3863007) qPCR

T--PuiChing_Macau--PCT.png

1b) PCT (BBa_K3863007) qPCR

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--result14.jpg

Figure 2b. IR spectrum of BBa_K3863004