Composite

Part:BBa_K3102043:Design

Designed by: Chloe DOIZELET   Group: iGEM19_Ionis_Paris   (2019-10-15)
Revision as of 18:27, 15 October 2019 by ChloeDOIZELET (Talk | contribs) (→‎Design Notes)


Lactate production (ldhA, lldP, ACS operon)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1478
    Illegal XhoI site found at 3404
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 489
    Illegal AgeI site found at 1614
    Illegal AgeI site found at 2077
    Illegal AgeI site found at 2515
    Illegal AgeI site found at 2674
    Illegal AgeI site found at 2686
    Illegal AgeI site found at 3833
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 219
    Illegal SapI.rc site found at 6698


Design Notes

In ACS operon, we mutated a Cytosine into Thymine at the 1563bp position (C1563T) because of the Esp3I enzyme that we used for our Golden Gate Assembly (GGA) construct. We also mutated an Adenine into Guanine and a Cytosine in Thymine at the 2425bp and 2428bp position (A2425G & C2428T), respectively, because of the EcoRI enzyme.

Source

LDHA, E.coli, sequence found through Ebi https://www.ebi.ac.uk/ena/data/view/AAC74462

LLDP, E.coli, sequence found through Trypanocyc http://vm-trypanocyc.toulouse.inra.fr/ECOLI/sequence?type=GENE&object=EG11961

ACS operon, sequence comes from E.coli and was found through Biocyc: https://biocyc.org/ECOLI/sequence-delimited?chromosome=COLI-K12&object=EG11448&up=0&down=2160&right=4287371&left=4283253&type=true


Promoter (BBa_I719005), RBS (BBa_B0034) and Terminator (BBa_B0015) are from the iGEM Registry.

References

Bunch PK, Mat-Jan F, Lee N, Clark DP. "The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli". Microbiology. 1997;143(1 997):187–95.

Dong JM et al., Three overlapping lct genes involved in L-lactate utilization by Escherichia coli., Journal of Bacteriology, 1993 Oct;175(20):6671-8.

Gimenez R et al., The Gene yjcG, Cotranscribed with the Gene acs, Encodes an Acetate Permease in Escherichia coli, Journal of Bacteriology, 2003 Nov; 185(21):6448-55.

Nunez MF et al., Transport of L-Lactate, D-Lactate, and glycolate by the LldP and GlcA membrane carriers of Escherichia coli, Biochem Biophys Res Commun. 2002 Jan 18;290(2):824-9.