Difference between revisions of "Part:BBa K3863006"

 
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<partinfo>BBa_K3863006 short</partinfo>
 
<partinfo>BBa_K3863006 short</partinfo>
  
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While an amylase ought to be capable of degrading starch, the product protein is believed to be periplasmic and thus ought to only degrade starch if it leaks from the periplasm in significant quantities. Its natural function inE. colipresumably involves degrading shorter glucose chains:Lengsfeldet al(2008)state that "MalS produces preferentially maltohexaose from longer maltodextrins in the periplasm".
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This is a composite part designed to break down starch in food waste into monosaccharides.
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Amylase: E. coli periplasmic α-amylase gene malS. The protein sequence in this part is based on K523001 from Edinburgh 2011.
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Glucoamylase: Caulobacter crescentus CB15, caulobacter vibrioides gene for glucoamylase(GenBank: AB813000)
  
 
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<partinfo>BBa_K3863006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3863006 SequenceAndFeatures</partinfo>
  
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==Team PuiChing_Macau 2021: Break down monosaccharides==
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===Validation of construct expression using RT-qPCR===
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https://2021.igem.org/wiki/images/7/7e/T--PuiChing_Macau--1aa.jpg
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<p>1a) Amylase (BBa_K3863006) qPCR</p>
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https://2021.igem.org/wiki/images/9/92/T--PuiChing_Macau--1b.jpg
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<p>1b) Glucoamylase (BBa_K3863006) qPCR</p>
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<p>Fig 1. Real-time qPCR fold change of reference gene</p>
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<p>
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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 K3863006 to the vector (pETDuet).
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</p>
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==Reference==
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<p>[1]Masayoshi Sakaguchi,corresponding author1 Yudai Matsushima,1 Toshiyuki Nankumo,1 Junichi Seino,1 Satoshi Miyakawa,1 Shotaro Honda,1 Yasusato Sugahara,1 Fumitaka Oyama,1 and Masao Kawakita1,2, (2014) (Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification)</p>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 15:50, 21 October 2021


a-amylase-HA-glucoamylase-his

This is a composite part designed to break down starch in food waste into monosaccharides. Amylase: E. coli periplasmic α-amylase gene malS. The protein sequence in this part is based on K523001 from Edinburgh 2011. Glucoamylase: Caulobacter crescentus CB15, caulobacter vibrioides gene for glucoamylase(GenBank: AB813000)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1287
    Illegal NotI site found at 2146
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 3884
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2441
    Illegal NgoMIV site found at 2633
    Illegal NgoMIV site found at 2637
    Illegal NgoMIV site found at 3275
    Illegal NgoMIV site found at 3649
    Illegal NgoMIV site found at 3803
    Illegal NgoMIV site found at 3824
    Illegal AgeI site found at 423
    Illegal AgeI site found at 1588
    Illegal AgeI site found at 2393
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3026

Team PuiChing_Macau 2021: Break down monosaccharides

Validation of construct expression using RT-qPCR

T--PuiChing_Macau--1aa.jpg

1a) Amylase (BBa_K3863006) qPCR

T--PuiChing_Macau--1b.jpg

1b) Glucoamylase (BBa_K3863006) 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 K3863006 to the vector (pETDuet).

Reference

[1]Masayoshi Sakaguchi,corresponding author1 Yudai Matsushima,1 Toshiyuki Nankumo,1 Junichi Seino,1 Satoshi Miyakawa,1 Shotaro Honda,1 Yasusato Sugahara,1 Fumitaka Oyama,1 and Masao Kawakita1,2, (2014) (Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification)