Part:BBa_K4213044
pNOS:TetR:tNOS
Engineering Cycle 2: Second Iteration: Establishing Repressor Expression
Design
Having learnt that the above-mentioned syntax works as intended, it was time to substitute the fluorescent protein with our repressor of choice. As already mentioned, the registry gave as the base sequence for TetR (BBa_C0040), but this part was intended for expression in prokaryotic organisms and not eukaryotic ones. For that reason, we needed to anticipate the presence of a nucleus, possible splicing sites and unoptimized expression on account of less frequently used synonymous codons present in the coding sequence (cds).
Tet Repressor for plants
Starting with the first, it seemed that due to the size of TetR (48 kDa) diffusion into the cell nucleus was more than likely [1]. Nevertheless, we decided to add a Nuclear Localization Signal (NLS), specifically Simian Virus 40 NLS [2], to the sequence since this would ensure its passage into the nucleus. Moving on to the possible splicing sites, the now NLS-containing sequence was run through a neural network based program called NetGene2. This method is proven to predict possible splicing sites within given sequences, based on analysis made on the plant Arabidopsis thaliana [3]. Analysis showed no potential splicing sites within the cds so we moved on with no modifications. Concluding with codon optimization, this is a tool revolving around the frequency that each synonymous codon occurs within a certain organism’s genome. We used IDT’s codon optimization tool for expression in Nicotiana benthamiana.
It is worth pointing out that, for the visualization of expression, we, again, decided to incorporate the modified mVenus into the sequence, leading to a construct consisting of pNOS:Venus:TetR:NLS:tNOS.
Build
Following the same steps as before and making the necessary optimizations to our protocols, needed to properly and efficiently get through the cloning procedures, we managed to confirm the assembly of the level alpha construct using diagnostic digestion.
Test
The testing of the produced construct was, again, done with model plant Nicotiana benthamiana by agroinfiltration. The sample was prepared with a special cell-permeant nuclear counterstain that emits blue fluorescence when bound to dsDNA (Hoechst 33342, Excitation λ: 350 nm, Emission λ: 460 nm) and can be distinguished from mVenus, when observed under both fluorescent microscopy and confocal microscopy, in order to confirm localization of our construct in the nucleus.
Learn
After examining our samples under the confocal microscope and taking our pictures we concluded that our transcriptional unit did manage to express mVenus:TetR:NLS in great numbers and the fused protein was mostly present within the nucleus, confirming the functionality of the NLS.
References
[1] Gatz, C., & Quail, P. H. (1988f, March). Tn10-encoded tet repressor can regulate an operator-containing plant promoter. Proceedings of the National Academy of Sciences, 85(5), 1394–1397. https://doi.org/10.1073/pnas.85.5.1394
[2] Lu, J., Wu, T., Zhang, B., Liu, S., Song, W., Qiao, J., & Ruan, H. (2021f, May 22). Types of nuclear localization signals and mechanisms of protein import into the nucleus. Cell Communication and Signaling, 19(1). https://doi.org/10.1186/s12964-021-00741-y
[3] Hebsgaard, S. (1996c, September 1). Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Research, 24(17), 3439–3452. https://doi.org/10.1093/nar/24.17.3439
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 192
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 944
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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