Difference between revisions of "Part:BBa K4339000"
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To determine the tolerance of BL21 DE3 pLysS to extracellular alanine, we produced growth curves for <em>E. coli</em> 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. | To determine the tolerance of BL21 DE3 pLysS to extracellular alanine, we produced growth curves for <em>E. coli</em> 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. | ||
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<!-- <center><img src="https://static.igem.wiki/teams/4339/wiki/alaninetolerance.jpg" alt="Growth curves for the control and CycA strains cultured in a range of extracellular alanine concentrations" style="width:1000px;height:475px;"></center> <!-- --> | <!-- <center><img src="https://static.igem.wiki/teams/4339/wiki/alaninetolerance.jpg" alt="Growth curves for the control and CycA strains cultured in a range of extracellular alanine concentrations" style="width:1000px;height:475px;"></center> <!-- --> | ||
Revision as of 13:30, 10 October 2022
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)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 340
- 1000COMPATIBLE 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.
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]