During our exploration of non-model organisms, we aimed to assess the efficacy of various standard biological components in facilitating protein expression within Acinetobacter baumannii and Klebsiella pneumoniae. This undertaking involved a combination of literature review and iterative experimentation to achieve our objectives.
BBa_J23119 expressing proteins in A. baumannii
Regarding the strong promoter BBa_J23119, both comprehensive literature exploration[1] and our empirical investigations confirm its capacity to drive protein expression within Acinetobacter baumannii. To evaluate this phenomenon, we conducted a fluorescence emission analysis employing the Thermofisher Scientific Varioskan Lux plate reader. This assessment focused on the signal of a reporter gene (i.e., from the BBa_I13507 iGEM part cloned downstream of the BBa_J23119 promoter), specifically the red fluorescent protein (RFP) with excitation at 535nm and emission at 610nm.
Since visual analysis of the colonies obtained after transfections showed different shades of red, our experimental setup involved four colonies of A. baumannii, each harboring the RFP reporter gene within a cassette integrated into our newly developed backbone, BBa_K4727000. To establish a comparative benchmark, we contrasted these expression levels with those of two iGEM constructs sharing the same RBS-RFP-terminator structure carried in our part (BBa_I13507), namely: a E. coli TOP10 strain expressing the RFP gene under the control of both the relatively weak promoter BBa_J23101 (the normalization strain proposed in Kelly et al.[2]) and the strong promoter BBa_R0040 (pTet).
Among the selected colonies, namely 1.2A, 1.2B, 1.1A, and 2.2A, a trio comprising 1.2A, 1.2B, and 1.1A exhibited analogous rates of reporter gene expression. This expression level was higher than the signal produced by BBa_J23101 promoter driving RFP; yet, it remained notably lower than the signal produced by a E.coli TOP10 strain bearing a pTet promoter driving RFP in the high copy plasmid pSB1A2 (iGEM part BBa_I13521). Conversely, colony 2.2A distinctly displayed heightened expression, comparable to the one shoved in E. coli by the I13521 cassette. Upon subjecting the expression cassettes to Sanger sequencing, we determined that the diminished fluorescence emission stems from a mutation within the RFP coding sequence. This mutation, rather than the promoter itself, leads to a significant reduction in emission intensity due to an amino acid substitution.
By elucidating the intricacies of this promoter-driven mechanism, our study sheds light on the potential of enhancing protein expression within non-model organisms, underscoring the significance of genetic components beyond promoters in governing gene expression dynamics.
BBa_J23119 expressing proteins K. pneumoniae
Given the versatility of our novel standard backbone, BBa_K4727000, which demonstrates efficacy in both A. baumannii and K. pneumoniae, we encountered the imperative need to identify a promoter that would function effectively in this secondary organism. Drawing from a combination of literature examination [3] and our own experimental findings, we ascertained that the same BBa_J23119 promoter could indeed operate proficiently in K. pneumoniae, exhibiting a strength comparable to its performance in A. baumannii.
The experiment was carried out in a similar fashion as described above: two colonies of K. pneumoniae were electroporated, following the protocol that we optimized, with part BBa_K4727000 expressing a reporter gene, namely RFP form the BBa_I13507, under the regulation of BBa_J23119 promoter. In parallel, the same wild type bacteria was used as a reference, together with our golden standards: E. coli expressing RFP under the control of pTet and J23101.
pTet (BBa_R0040) expressing proteins in A. baumannii
We then decided to attempt the cloning of an expression cassette bearing the pTet promoter (BBa_R0040) driving RFP (final construct, BBa_I13521) inside our plasmid backbone for A. baumannii and K. pneumoniae BBa_K4727000. Once successful cloning was achieved, we tried to electroporate the assembled construct in A. baumannii, using our optimised protocol. After the transformation, some red colonies could be seen; so, as done before, a plate reader experiment was carried out to assess the ability of the pTet promoter to allow protein expression in this bacteria.
From the resulting data we can assess that BBa_R0040 can express proteins in A. baumannii at a level significantly lower than the one shown by the same promoter in E. coli, but higher than the weak promoter J23101. The shown expression level could be compared to the one of BBa_J23119 in A. baumannii. This result was of paramount importance to design our expression cassettes for the dCas and our designed guides, to allow for expression of both the sequences.
BBa_B0010 & BBa_B0012 terminating transcription in A. baumannii and K. pneumoniae
In the previous experiments, as described above, the expression cassettes were completed with a double terminator composed of parts BBa_B0010 and BBa_B0012. With the results presented here we can assess how these sequences can act as terminators in the non-model organisms A. baumannii and K. pneumoniae.
E1010 as a reporter gene in A. baumannii and K. pneumoniae
While testing our expression cassettes and to assess the capability of our newly introduced plasmid backbones in non model organisms, we needed a suitable reporter gene. In this context we thought of using the part BBa_E1010, as it represents the most commonly used gene reporter in E. coli. This part, as demonstrated above, proved to be suitable for the intended purpose in both the tested species.
References
- Wang et al., “A Highly Efficient CRISPR-Cas9-Based Genome Engineering Platform in Acinetobacter baumannii to Understand the H2O2-Sensing Mechanism of OxyR”, Cell Chemical Biology 26, 1732–1742 December 19, 2019
- Kelly, J.R., Rubin, A.J., Davis, J.H. et al. Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng 3, 4 (2009). https://doi.org/10.1186/1754-1611-3-4
- Y. Wang et al., ‘CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae’, Appl. Environ. Microbiol., vol. 84, no. 23, pp. e01834-18, Nov. 2018, doi: 10.1128/AEM.01834-18.