Difference between revisions of "Part:BBa K4339000"

Revision as of 13:43, 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)

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.

Growth curves for CycA and control strains in a range of alanine concentrations, plotted using OD₆₀₀ measurements

Growth of control vs CycA strains

To characterise the effect of CycA inclusion, we produced growth curves for both strains in blank and alanine-doped LB, measuring cells/ml for each sample every hour via image flow cytometry using an Imagestream. Figure 2 below shows the variation in cells/ml for each condition with time. It is apparent that the form of growth is essentially the same for the control strain at both alanine concentrations, having similar final cells/ml values and entering the log and stationary phases of growth at similar points in time (t = 3 and 6 hours after inoculation respectively).

The growth for the CycA strain seems to vary with alanine concentration. In undoped LB, the CycA strain does not appear to reach stationary phase within the time range measured over, suggesting expression of the protein has delayed the onset of this phase of growth. Whereas, in doped LB, the CycA strain appears to reach stationary phase at an earlier time point than the other conditions (t = 5 hours after inoculation) and may in fact enter death phase, with the final cells/ml dropping from t = 6 to t = 7 hours after inoculation. It is possible that the combination of expression of (as we will later see) likely non-functional and misfolded CycA proteins as inclusion bodies, and the associated metabolic strain is sufficient to trigger cell death by this time point.

Growth curves for CycA and control strains in blank and alanine-doped LB plotted using image flow cytometry measurements

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]