Part:BBa_K3110013
Strong Promoter Strong RBS lldR+lldD
This construct has L-Lactate Dehydrogenase (lldD) and Regulator element (lldR) under the control of a strong promoter and a strong RBS. lldD cleaves L-Lactate to pyruvate and lldR has the dual role of an activator and a repressor. Our intention of using this part is to have control over the detection threshold of L-Lactate by the lldPRD regulatory region. We have also designed other parts which produce various lower concentrations of lldD and lldR by varying the strength of the promoter and RBS controlling their production.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1988
Illegal AgeI site found at 620 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1425
Characterization
During the course of our experiments, we had some technical difficulties with SOEing, but we got the constructs after a few attempts. The SOEed construct was digested and ligated to the plasmid and transformed into DH5alpha, but no positive colonies were obtained on colony PCR. We could not pursue this further due to time constraints, hence the above construct was not characterised. The other constructs that were designed by varying the strengths of RBS and promoter for this lldR + lldD gene combination were not SOEed as we could not standardise the protocols.
Design
Strong promoter -Strong RBS-lldR flank and lldD flank-terminator were synthesized by IDT
lldR+lldD was amplified from the genome
Reference
iGEM ETH zurich 2015
iGEM NUS Singapore 2016
Aguilera, L., Campos, E., Gimenez, R., Badia, J., Aguilar, J., & Baldoma, L. (2008). Dual Role of LldR in Regulation of the lldPRD Operon, Involved in L-Lactate Metabolism in Escherichia coli. Journal of Bacteriology, 190(8), 2997–3005. doi:10.1128/jb.02013-07
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