To evaluate and compare surface display efficiency across species, E. coli was used as a benchmark chassis alongside Caulobacter crescentus and Synechococcus elongatus UTEX 2973. Using an established construct design from the distribution kit, we performed transformations and validation experiments to assess expression and functionality of the surface display system under standardized conditions.
Introduction - Benchmark
To validate the CA displayed on our transformed CB2A, we required a benchmark for surface display efficiency and enzymatic activity. As such, we transformed the BL21 strain of E. coli with a literature-validated CA display vector, adapted from [1]. While E. coli does not naturally have a surface-layer, we utilized the N-terminal domain of the ice nucleation protein to export and anchor our CA candidates to the outer membrane.
Construct Design
This construct is inspired by the vector described in a previous study using ice nucleation proteins to display carbonic anhydrases in E. coli[1]. While previous literature uses the pET-22b(+) plasmid, we made assembly of this construct more accessible for other teams by utilizing the cloning vector BBa_J435300 (referred to as pDest) from the iGEM distribution kit to functionally resemble pET-22b(+). This modified plasmid was formed through Golden Gate assembly with BsaI. The first backbone feature we modified was the removal of illegal SapI site. Secondly, we supplemented the original AmpR antibiotic resistance cassette with the chloramphenicol resistance cassette to match the selection system used for CB2A. Thirdly, we introduced a modified MCS similar to that used in the CB2A constructs, which is flanked by SapI recognition sites appended with additional homologous ends. The fourth modification was removing the inducible lacI promoter and gene, then replacing it with a constitutive J23100 promoter. Finally, we inserted the INPN-Re fusion sequence for surface display and incorporated a Myc tag for detection.
E. coli display plasmid diagram
Transformation
We were able to successfully assemble the recombinant display backbone, verified by sequencing results. Several CAs were successfully cloned, validated by colony PCR and restriction digest. We found that our bacteria took 1 - 2 days longer than expected to yield clonies after heat shock transformation, which could be due to the addition of chloramphenicol selection cassette. Due to time constraints, we were only able to clone BtCAII into BL21 for the display strain.
1. Zhu Y, Liu Y, Ai M, Jia X. Surface display of carbonic anhydrase on EscherichiaColi for CO2 capture and mineralization. Synthetic and Systems Biotechnology [Internet]. 2022 Mar 1 [cited 2025 Feb 19];7(1):460—73. Available from: https://www.sciencedirect.com/science/article/pii/S2405805X21000764
