Composite

Part:BBa_K2762012:Design

Designed by: Chen-Wei Chen   Group: iGEM18_NCKU_Tainan   (2018-09-29)
Revision as of 07:05, 29 September 2018 by Phixdvd (Talk | contribs) (→‎Design Notes)


PlacI-B0034-rbcL-B0015-PlacI-B0034-rbcX-B0034-rbcS-B0015


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 1375
    Illegal AgeI site found at 478
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

1 Usage: 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 and then form two 3-phosphoglycerate molecular. 3-phophoglycerate will then convert to pyruvate by the native metabolic system of E coli. Previous studies have utilized E. coli as a host of studying point mutation and random mutation of Rubisco in order to enhance its enzyme activity. Which proves the concept of installing rubisco into E. coli feasible. After mining information from journal, we selected rubisco form Synechococcus elongtus PCC. 7002, which is a well studied cyanobacteria.

2 Biology: The structure of RubisCo involves two polypeptide subunit: RbcL and RbcS. Each RubisCo is consist of eight RbcL and RbcS. RbcL, the large chain of RubisCo, contain the main active site. The active site requires Mg2+ ion. The ion binds to the certain amino acid of the centrol of RbcL, activate the RubisCo. Since the M9 medium contains Mg2+ ion, we only need to add little amount of additional ion, adjusting the concentration to 20mM.

It has reported that rbcL can be functional solely. However, researches have revealed that RbcS contribute to the activity and the CO2/O2 specification through the mutagenesis and hybridization of cynobacteria RubisCo, and indicated the RbcS should take into consideration while constructing Calvin cycle. For this reason, we decide to add RbcS gene into our construction.

RbcX, the subunit which doesn’t involve in the structure of RubisCo, however, plays an important role of the structure folding . Researches have revealed that without the presence of the RbcX chaperon, RubisCo cannot folding correctly. According to these researches, we take the RbcX into our consideration and add the gene into our construction.

3 Characterization: We did three experiments to confirm the expression and function of this part. First we inserted the part on pSB1C3 and cloned the plasmid into DH5 alpha. We extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. Second, we did the SDS PAGE to confirm the present of each polypeptide. Third, we cultured the W3110 with the the PLacI-RubisCo part and PRK part in M9-xylose medium in 5% CO2 incubator and normal incubator after inducing the RubisCo gene with IPTG. We also did the non-induced control groups. The result showed that the with the present of these two functional enzyme. The E. coli grew faster and consumed more xylose in the CO2 rich environment.

Source

Synechococcus elongtus PCC. 7002. The sequence is codon optimized for larger expression in E. coli.

References