Difference between revisions of "Part:BBa K2762000:Design"

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===Design Notes===
 
===Design Notes===
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We first codon optimized the <i>prk</i> sequence and sent it to IDT for gene synthesis. The gene fragment is amplified via PCR reaction for cloning. The sequence is then cloned into the pSB1C3 plasmid with HindIII and SpeI.
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1 Usage:
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Ribulose-1,5-biphosphate carboxylase/oxygenase catalyzes the first reaction of the Calvin cycle, converting the combination of Ribulose-1,5-biphosphate(RuBP) and carbon dioxide to two 3-phosphoglycerate molecular. The rbcL part encodes the large subunit of the RubisCo enzyme, which also contain the active site of the enzyme. In our carbon fixing  pathway, the RubisCo enzyme is the most important enzyme catalyzing the reaction of RuBP and CO2.
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2  Biology:
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The rbcL gene is from  cynobacteria <i>Synechococcus elongatus PCC 7002</i>.The activation of RubisCo will be maximize with the binding of the rbcS subunit. To get more information, see our  composite part: RubisCo
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3 Characterization:
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We did two experiment to confirm the expression of this part. First we inserted the part on pSB1C3 and transformed the plasmid into DH5 alpha. We extracted the plasmid after the the formation of the colony and screened the consruction by enzyme digestion. Second, we cloned the plasmid into BL21(DE3) and induced the promoter with IPTG. We than did the SDS-PAGE to confirm the present of the RbcL.
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===Source===
 
===Source===

Revision as of 08:25, 29 September 2018


rbcL (Large subunit of the ribulose-bisphosphate carboxylase/oxygenase)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1149
    Illegal AgeI site found at 252
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We first codon optimized the prk sequence and sent it to IDT for gene synthesis. The gene fragment is amplified via PCR reaction for cloning. The sequence is then cloned into the pSB1C3 plasmid with HindIII and SpeI.

Source

Codon oprimized Synechococcus elongatus PCC7002

References