Part:BBa_K2941001
GFP-TAG-RFP (improved from BBa_K567018)
Intro
This part is improved from the biobrick BBa_K567018 by removing the T7 promoter sequence. Since this part lacks the T7 promoter sequence, the biobrick can be directly used to test incorporation of noncanonical amino acids (ncaa) in amber stop codons in Escherichia coli hosts lacking T7 like the amberless C321.deltaA.exp (321.A) strain. The original iGEM team, STJU-BioX Shanghai have explained, that the idea of this part was to be able to report the functionality of their tRNA synthetases. We used it to optimize our production.
What is it and how should it work?
The part consists of green fluorescent protein (GFP) and red fluorescent protein (RFP) connected by 11 amino acids long glysine-serine linker with an amber stop codon in between. Only GFP would be present, when the fluorescent reporter is expressed. Ideally RFP would be present only, when a tRNA synthetase with ncAA recognizing TAG codons is expressed. Theoretically the strain we used would not have been able to express even GFP without the tRNA synthetase due to the absence of the release factor 1 (RF1).
How did it work in our experiments?
Materials & Methods
We cloned this in pUC19 vector, which we found to have a bit leaky expression under the lac promoter. The plasmid was then co-transformed into 321.A, which was precultured before dispensing it 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).
Results & Discussion
We reached our goal to get more measurement data on the function of the reporter, 321.A and the tRNA synthetase. With our test setting we were actually able to detect previously undocumented RFP from the reporter. When the GFP and RFP signals were further compared using this fluorescence change (increase of GFP / increase of RFP), which is represented in a 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.
Here we have presented how the removal of the T7 binding site broadens up opportunities for its applications.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1302
Illegal AgeI site found at 1414 - 1000COMPATIBLE WITH RFC[1000]
| None |