Difference between revisions of "Part:BBa K3114008"
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===Usage and Biology===
 
===Usage and Biology===
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BBa_K3114008 is a golden gate compatible part which allows insertion of the NYC1 gene into genetic circuits to express the enzyme chlorophyll B reductase.
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BBa_K3114008 is a golden gate compatible part which allows insertion of the NYC1 gene into genetic circuits to express the enzyme chlorophyll B reductase. In creating this part, iGEM Calgary wished to allow the golden gate cloning of this plant enzyme into <i>E.coli</i> for other teams to use in projects involving degradation of chlorophyll. This part was designed to be golden-gate compatible based on the Mo-Clo standard (Weber et al., 2011). It is also compatible with the BioBrick RFC[10] standard.
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===Design===
 +
When designing this part and the rest of our collection, we were interested in creating parts that could be used in Golden Gate assembly right out of the distribution kit without the need to first domesticate them in a Golden Gate entry vector. As such, these parts are not compatible with the iGEM Type IIS RFC[1000] assembly standard because we included the BsaI restriction site and MoClo standard fusion site in the part’s sequence.
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As per the MoClo standard, the 5’ signal peptide fusion sequence included in this part is AATG, and the 5’ signal peptide fusion sequence is AGGT.
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[[Image:T--Calgary--MoClo1.png|900px|thumb|center|Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).]]
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This part includes a start codon. A Gly-Gly spacer sequence was added to the end of the signal peptide sequence in order to ensure that the fused protein of interest will be in-frame following Golden Gate assembly.
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This sequence has been codon optimized for high expression in <i>E. coli</i>.
  
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==Collection of Parts==
 
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This part was designed to be Golden Gate compatible with certain Golden Gate parts in iGEM Calgary's 2019 part collection including the following: <br/>
 
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1. DsbA signal peptide - Bba_K3114000 https://parts.igem.org/Part:BBa_K3114000<br/>
 
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2. MalE signal peptide - Bba_K3114001 https://parts.igem.org/Part:BBa_K3114001<br/>
 
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3. OmpA signal peptide - Bba_K3114002 https://parts.igem.org/Part:BBa_K3114002<br/>
 
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4. PhoA signal peptide - Bba_K3114003 https://parts.igem.org/Part:BBa_K3114003<br/>
 
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5. Tat signal peptide  - Bba_K3114004 https://parts.igem.org/Part:BBa_K3114004<br/>
 
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6. TorA signal peptide - Bba_K3114005 https://parts.igem.org/Part:BBa_K3114005<br/>
 
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7. Double Terminator  - Bba_K3114013 https://parts.igem.org/Part:BBa_K3114013
 
  
  
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Horie, Y., Ito, H., Kusaba, M., Tanaka, R., & Tanaka, A. (2009). Participation of chlorophyll b reductase in the initial step of the degradation of light-harvesting chlorophyll a/b-protein complexes in Arabidopsis. Journal of Biological Chemistry, 284(26), 17449-17456.
 
Horie, Y., Ito, H., Kusaba, M., Tanaka, R., & Tanaka, A. (2009). Participation of chlorophyll b reductase in the initial step of the degradation of light-harvesting chlorophyll a/b-protein complexes in Arabidopsis. Journal of Biological Chemistry, 284(26), 17449-17456.
  
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Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. <i>PLoS ONE</i>, 6(2). https://doi.org/10.1371/journal.pone.0016765
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3114008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3114008 SequenceAndFeatures</partinfo>

Revision as of 23:25, 18 October 2019


Chlorophyll B Reductase (CBR)

Chlorophyll B Reductase is a naturally occurring plant enzyme, encoded by the gene NYC1, which catalyzes the first step of chlorophyll b degradation (Horie et al. 2009). With the cofactor NADPH, chlorophyll b reductase converts chlorophyll b into 7-Hydroxymethyl Chlorophyll a.


Usage and Biology

BBa_K3114008 is a golden gate compatible part which allows insertion of the NYC1 gene into genetic circuits to express the enzyme chlorophyll B reductase. In creating this part, iGEM Calgary wished to allow the golden gate cloning of this plant enzyme into E.coli for other teams to use in projects involving degradation of chlorophyll. This part was designed to be golden-gate compatible based on the Mo-Clo standard (Weber et al., 2011). It is also compatible with the BioBrick RFC[10] standard.

Design

When designing this part and the rest of our collection, we were interested in creating parts that could be used in Golden Gate assembly right out of the distribution kit without the need to first domesticate them in a Golden Gate entry vector. As such, these parts are not compatible with the iGEM Type IIS RFC[1000] assembly standard because we included the BsaI restriction site and MoClo standard fusion site in the part’s sequence.

As per the MoClo standard, the 5’ signal peptide fusion sequence included in this part is AATG, and the 5’ signal peptide fusion sequence is AGGT.

Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).

This part includes a start codon. A Gly-Gly spacer sequence was added to the end of the signal peptide sequence in order to ensure that the fused protein of interest will be in-frame following Golden Gate assembly.

This sequence has been codon optimized for high expression in E. coli.


References

Horie, Y., Ito, H., Kusaba, M., Tanaka, R., & Tanaka, A. (2009). Participation of chlorophyll b reductase in the initial step of the degradation of light-harvesting chlorophyll a/b-protein complexes in Arabidopsis. Journal of Biological Chemistry, 284(26), 17449-17456.


Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0016765


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 4
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 1061