Coding
CycA

Part:BBa_K4339000

Designed by: Grace Poulton   Group: iGEM22_Exeter   (2022-07-06)
Revision as of 13:23, 10 October 2022 by Matthewludwig (Talk | contribs)


Non-polar amino acid transporter (CycA)

CycA is an inner membrane permease [1], which can facilitate the uptake of non-polar amino acids (including D-alanine, D-serine, glycine and L-alanine) via a proton symport mechanism [2]. CycA was first identified in the K12 strain of E. coli.

CycA was codon-optimised for expression in E. coli BL21 DE3 strains and their derivatives. CycA was fused with the constitutive promoter PJ23100 and a C-terminus His tag and engineered into the strain BL21 DE3 pLysS. CycA was engineered into this strain with an aim to increase uptake of L-alanine from an alanine-doped LB growth medium, to facilitate synthesis of alanine-rich proteins such as MaSps (Major Ampullate Silk proteins).


Usage and Biology

Sequence and Features

The sequence originates from E. coli MG1655 K-12 (Hook et al. 2022)


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 AgeI site found at 340
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterisation

Alanine tolerance

To determine the tolerance of BL21 DE3 pLysS to extracellular alanine, we produced growth curves for E. coli engineered with either an empty pX1900 plasmid backbone (control strain), or the cycA gene present in this backbone (CycA strain), incubated in a range of alanine concentrations from 5.8 (blank LB) up to 100 mM. As is indicated below in figure 1, there is no obvious difference in the growth across the range of extracellular alanine concentrations studied, for both strains examined, suggesting both can tolerate extracellular alanine concentrations up to 100 mM.

<img src="alaninetolerance.jpg" alt="Growth curves for the control and CycA strains cultured in a range of extracellular alanine concentrations" style="width:1000px;height:475px;">


References

[1] - Hook C et al. The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production. Microorganisms. 2022;10(3):647. doi: https://doi.org/10.3390/microorganisms10030647

[2] - CycA. ECOCYC version 26.0. Available at: https://biocyc.org/gene?orgid=ECOLI&id=EG12504 [Accessed 14/9/22]

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Categories
Parameters
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