Difference between revisions of "Part:BBa K3110014"
 
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<h1>Characterization </h1>
 
<h1>Characterization </h1>
 
During the course of our experiments, we had some technical difficulties with SOEing, but we got the first construct for this combination (Strong Promoter Strong RBS lldR lldD) after a few attempts. That 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 it further due to time constraints, hence the first construct was not characterised. This particular construct was designed by varying the strengths of RBS and promoter for the same lldR + lldD gene combination, but was not SOEed and characterised as we could not standardise the protocols.
 
During the course of our experiments, we had some technical difficulties with SOEing, but we got the first construct for this combination (Strong Promoter Strong RBS lldR lldD) after a few attempts. That 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 it further due to time constraints, hence the first construct was not characterised. This particular construct was designed by varying the strengths of RBS and promoter for the same lldR + lldD gene combination, but was not SOEed and characterised as we could not standardise the protocols.
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<h1>Design</h1>
 
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Strong promoter-Medium RBS-lldR flank and lldD flank-terminator were synthesized by IDT <br>
 
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lldR+lldD was amplified from the genome
 
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<h1>Reference</h1>
 
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iGEM ETH zurich 2015<br>
 
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iGEM NUS Singapore 2016<br>
 
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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
 

Latest revision as of 14:52, 21 October 2019


Strong Promoter Medium RBS lldR+lldD

This construct has L-Lactate Dehydrogenase (lldD) and Regulator element (lldR) under the control of a strong promoter and a medium 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 higher and lower concentrations of lldD and lldR by varying the strength of the promoter and RBS controlling their production.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1989
    Illegal AgeI site found at 621
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1426


Characterization

During the course of our experiments, we had some technical difficulties with SOEing, but we got the first construct for this combination (Strong Promoter Strong RBS lldR lldD) after a few attempts. That 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 it further due to time constraints, hence the first construct was not characterised. This particular construct was designed by varying the strengths of RBS and promoter for the same lldR + lldD gene combination, but was not SOEed and characterised as we could not standardise the protocols.