Team:Aboa/Contribution
Characterization and Contribution
BBa_K567018 (T7-GFP-TAG-RFP)
We decided to use the biobrick BBa_K567018 as our contribution as it was designed to report the functionality of tRNA synthetases adding amino acids to amber stop codon (TAG). (1). However, we did not find documentation on how to produce and measure the reporter. Therefore, we decided to test it ourselves to investigate how it works in systems such as the one we used.
The biobrick was retrieved from the 2019 distribution kit's second plate, well O1. The reporter fusion protein was under T7 binding site, but unfortunately our amberless host strain Eschericia coli C321.deltaA.exp (321.A) lacked the T7 RNA polymerase, and therefore it could not produce the reporter. However, 321.A was used to produce the antibodies, because the strain is specifically modified to efficiently incorporate non-canonical amino acids to TAG codon (2).
To overcome this problem, we decided to digest the biobrick with EcoRI to clone the GFP-TAG-RFP gene in pUC19 vector. However, as the digested fragments were separated on agarose gel, it was found out that all of the digestion fragments and the undigested residue were 500 bp longer than they were supposed to be, while the other plasmids on the same gel cut with the same enzyme were the size they were supposed to be. The digestion and gel electrophoresis were repeated with the same results (Figure 1). We might have virtually constructed the plasmid wrong, but the DNA band close to 2000 bp was still cut out and purified.
Figure 1. The gel separation of EcoRI digested pUC19 and BBa_K567018, 1 kb Generuler was used as a ladder. In both gel runs the linearized pUC19 (samples 1 and 3) was near the 2500 bp ladder band, where it should have been (2686 bp real size). The GFP-TAG-RFP insert, which was supposed to be 1569 bp, was in the first run (sample 2) and in the second run (sample 4) near the 2 kb standard band.
The first construct did not work. The cells did express the antibiotic resistance from the vector, but no glowing colonies were detected, which is why we decided to just order the GFP-TAG-RFP sequence from IDT.
After successful ligation of the synthetized GFP-TAG-RFP fragment into the pUC19 vector and transforming it into E. Coli, XL-1 Blue (XL-1) glowing colonies were detectable from the plate without isopropyl beeta-D-1-thiogalactopyranoside (IPTG) in the absence of glucose (Figure 2).
Figure 2. The XL-1 with the GFP-TAG-RFP synthetized fragment ligated into pUC19. On both of the plates there was ampicillin without IPTG, but the plate on the right also had glucose, which improves the inhibition of lac promoter as seen in the above. The glowing colonies were obtained, which indicated a successful production of GFP the least.
The reporter was tested in a well documented and functional host with pEVOL-pAzF plasmid (3) that codes for a tRNA synthetase for p-azido-L-phenylalanine (pAzF) recognizing TAG. After preculturing, 200 µL of growth was dispensed per well on a 96-well plate, after which we added different amounts of inducers IPTG and arabinose as well as pAzF at OD of 0.5. The growths were incubated the cultures in Cytation 5 over night by shaking the plate in 30 °C. Fluorescence signal (RFU) of GFP and RFP was measured with the preset filters of the Cytation 5 at 60 min intervals throughout the incubation.
Because fluorescence signals were already high at the moment of the inducement, 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).
Our goal to get more measurement data on the reporter was reached. 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 (relative change of GFP / relative change of RFP), which is represented in a figure below. From the figure 3 it can be 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.
Figure 3. The 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 into the protein. Increase of arabinose concentration however decreased the ratio, and at 0,5 % arabinose, there is not that much difference in the effect of IPTG concentration.
However, because of the removal of T7 RNA polymerase binding site and the leaky expression vector, we cannot say for sure, that the original part would work for the same concentrations, strain, tRNA synthetase etc. However, based on our measurement data, RFP could be functional in the reporter, even though it we were not able to detect it visually. Moreover, because the removal of T7 binding site broaden 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].
BBa_R0010 (LacI regulated promoter)
E.coli XL-1 blue and 321.deltaA lack T7 RNA polymerase so tests with BBa_K567018 needed to be redesigned. You can read about this redesign process from Improve. This improved part was decided to insert under BBa_R0010, lacI regulated promoter, so it could be characterized during this project.
GFP-TAG-RFP part of the BBa_K567018 was ordered from iGEM sponsor offering gene synthetis. Gene was inserted into a pUC19-plasmid under BBa_R0010, lacI regulated promoter. The ready plasmid was inserted into E.coli 321.deltaA with another, p-azido-L-phenylalanine (pAzF) tRNA synthetase gene containing, plasmid (pEVOL-pAzF) to verify the strain's capability to suppress TAG-stop codon, incorporate new amino acid instead stop and continue translation as if it is just an regular amino acid.
GFP-TAG-RFP production was tested with isopropyl beeta-D-1-thiogalactopyranoside (IPTG) concentrations of 0 mM, 0.5 mM and 1mM, arabinose (pEVOL-pAzF is under arabonose promoter) concentrations of 0, 0.2 %, 0.5 %, 1 % and pAzF concentrations of 0 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1mM, 1.5 mM, 2 mM and 3 mM. Growths were started on black, clear bottom 96 well plate with 200 µL of 0.1 OD pregrowth in each well with SB medium using ampicillin, cloramphenicol and zeocin antibiotics. Growths were incubated for 2h in 37 °C before adding arabinose and pAzF and 10 min from that IPTG was added.
Growths were incubated in Cytation5 overnight in 37 °C shaking. Production rate of GFP-TAG-RFP was measured with Cytation5 GFP (excitation: 395 nm, emission: 509 nm)and mCherry (excitation 570 nm, emission: 615 nm) programs. Relative fluorescence is calculated as fluorescence at the start of induction with IPTG, pAzF and arabinose divided by the end of incubation the next morning. The relative change of RFP fluorescence divided by the change of GFP fluorescence after induction with variable amounts of p-azido-l-phenylalanine, IPTG and arabinose. Relative change of fluorescence is used to normalize signals that are present before induction.
Results from whole work is presented at Figure 1.
Figure 1. 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 into the protein. Increase of arabinose concentration however caused decrease in the ratio, and at 0,5 % arabinose, there is not that much difference in the effect of IPTG concentration.
And results about effects of LacI promoter is presented at Figure 2.
Figure 2. Relative signal increases of GFP and RFP. IPTG cocentration has little or no effect on relative signal increase.
As we can see from Figure 2, IPTG has little or no effect on GFP-TAG-RFP expression rate. Arabinose and pAzF are more cruciable for GFP-TAG-RFP expression as we can see from Figure 1.
To conclude this, BBa_R0010 LacI promoter is quite leaky and should not be used when strict expression control is required.