Team:Aboa/Improve
Improving a previous biobrick
We first started to work with the biobrick BBa_K567018, because it was designed for validating the functionality of tRNA synthetases adding amino acids to the amber stop codon (TAG) (1). The biobrick is a reporter fusion protein, where GFP and RFP are connected via a linker, that has an amber stop codon (TAG), in the middle of it. In the absence of a tRNA synthetase and its appropriate amino acid, the translation would stop at TAG. Therefore, it appeared to be a great tool for testing the incorporation of our non-canoninal amino acid (ncAA), p-azido-L-phenylalanine (pAzF) in TAG codons.
However, the reporter fusion protein was under a T7 RNA polymerase binding site, and unfortunately our amberless host strain Eschericia coli C321.deltaA.exp (321.A) lacked the T7 RNA polymerase. We still had to use 321.A to produce our antibodies, because the strain is specifically modified to efficiently incorporate ncAAs in TAG codon (2).
To overcome the lack of the T7 RNA polymerase, we decided to digest the biobrick with EcoRI to clone the GFP-TAG-RFP gene to the pUC19 vector.
Contents
BBa_K567018 (T7-GFP-TAG-RFP)
We ordered the GFP-TAG-RFP sequence of the biobrick from IDT and cloned it into pUC19, where the reporter was under the lac promoter. We transformed the plasmid, along with the pEVOL-pAzF plasmid, (3) coding for a tRNA synthetase for p-azido-L-phenylalanine (pAzF) recognizing TAG, into 321.A. We precultured the cells before dispensing 200 µl of growth per well on a 96 well plate.
When the OD reached 0.5, different amounts of inducers, IPTG and arabinose as well as pAzF were added. We then incubated the cultures in Cytation 5 over night by shaking the plate in 30 degrees. The fluorescence signal (RFU) of GFP and RFP were measured with the preset filters of the Cytation 5 at 60 min intervals throughout the incubation. For GFP, the preset filter’s excitation wavelenght was 395 nm and emission 509 nm, while for RFP they were 570 nm and 615 nm respectively.
Because the fluorescence signals were already high at the moment of the inducement, we used the fluorescence relative change was used to analyze results (as in relative change = fluorescence at the end / fluorescence at the moment of inducement). The GFP and RFP signals were further compared using this fluorescence change (relative change of GFP / relative change of RFP).
We reached our goal to get more measurement data on the reporter. With our test setting, when the GFP and RFP signals were further compared using the fluorescence change (increase of GFP / increase of RFP), we were actually able to detect previously undocumented RFP from the reporter. The results are represented in the figure below.
Figure 3. Ratio of relative RFP signal to the relative GFP signal at different concentrations of IPTG, arabinose and pAzF. Overall the increase of pAzF also increased the ratio, which indicates incorporation of the ncAA. Increase of arabinose concentration however caused decreased the ratio, and at 0,5 % arabinose, there is not that much difference in the effect of IPTG concentration.
From the figure 3 we concluded, that our system was leaky and that in the absence or at low concentrations of pAzF and arabinose, there might be something else incorporating into the fusion protein. However, the relative signal ratio was pretty much the same at 0.5 % and 1 % of arabinose, which is probably the point, where the pAzF-tRNA production is optimal.
Because the removal of the T7 binding site broadens up opportunities for its applications, we would like to consider this fragment as a new improved part.
1. STJU-BioX Shanghai team 2011. Biobrick BBa_K567018. [html visited 05/08(2019].
2. Escherichia coli C321.deltaA.exp from Addgene. [html document visited 10/09/2019].
3. pEVOL-pAzF plasmid from Addgene. [html document visited 10/12/2019].