Revision as of 04:04, 15 October 2019

PT7-GFP-TAG-RFP

GFP and RFP linked with a flexible chain and a stop codon TAG is inserted in the flexible chain. This biobrick is under the control of T7 promoter and lac operator. This part is used to testify the function of PT7-TDRS (BBa_K567011) and tRNA(Asp)-TAG (BBa_K567013).

Usage and Biology

Figure 1. A typical expression profile of pT7-GFP-TAG-RFP in NiCo21(DE3) cells. (Note that error bars are only shown for the negative control and 0.2mM IPTG induction; however, the error bars of the other samples are comparable to the 0.2mM IPTG curve. Each sample was tested with 6 replicates.) IPTG induction of the cells lifts the repression of lacUV5 promoter controlling the expression of T7 RNA Polymerase. The GFP in this part can then be transcribed and translated. Note that NiCo21 (DE3) cells do not contain an amber suppressor, and so will not read through the TAG stop codon. No RFP fluorescence was detected as expected (data not shown). The concentrations of IPTG were chosen based on the recommendation that BL21 (DE3) cells and derivatives be induced with IPTG concentrations between 0.5mM and 1mM. As seen from the graph, during the first two hours, 0.2 mM IPTG induced the cells equally as well as did 1mM IPTG. However, at 0mM IPTG there was significant leaky expression of GFP. This is likely due to the fact that this part contains a lacI binding site. The large copy number of pSB1C3 causes all the repressor to bind the part, rather than the lacUV5 controlling the expression of T7 RNAP. As such, significant amounts of T7 RNAP is present in the cells, which causes leaky expression of the part. A similar part that lacks the lacI binding site does not exhibit any leaky expression (see the graph here).


Figure 2.Plates with E. coli containing this part visualized under blue light with an orange filter. The left plate contains DH5a cells, whereas the right plate contains NiCo21(DE3) cells, not induced with IPTG. As seen from this picture, even with no IPTG induction, this part causes cells with the DE3 lysogen to fluoresce strongly.

Contributed by the Pitt 2015 iGEM team.

Optimizing protein production in expanded genetic code

Expanded genetic code is a technique where one of an organism’s codons, for example a stop-codon, is reprogrammed to code for a non-canonnical amino acid (ncAA) 1.

This includes replacing a desired codon throughout the genome with an analogous codon and adding an aminoacyl-tRNA synthetase recognizing the replaced codon to be able to introduce a ncAA instead 1.

We used the part to optimize our productions, because of the TAG stop codon between the two fluorescent proteins. Ideally RFP would be present only, when a tRNA synthetase with ncAA recognizing TAG codons is expressed. Also 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)2.

In our work we used Escherichia coli C321.deltaA.exp (321.A) strain, which has all its amber stop codons replaced and RF1 removed to make it possible to incorporate a ncAA in amber codons2.. However, because the strains lacks T7 RNA polymerase it could not have been able to produce the reporter, which is why we ordered just the GFP-TAG-RFP fragment of it. With this we were able to produce the reporter protein. At least we detected GFP and RFP with Cytation 5 fluorometry by exciting GFP at 395 nm and RFP at 570 nm wavelength, after which the emissions were measured at 509 nm and 615 nm respectively.

We precultured 321.A containing pEVOL-pAzF (3) expressing for tRNA synthetase for p-azido-L-phenylalanine (pAzF) recognizing TAG and GFP-TAG-RFP in pUC19 before dispensing 200 µl per well on a 96 well plate. When the OD of 0,5 was reached we added different amounts of inducers IPTG and arabinose as well as pAzF. Cultures were shaken as previously at 30 degrees. Fluorescence signal (RFU) was followed at GFP and RFP preset filters at 60 min intervals over night in Cytation 5.

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). Then 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 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.

Figure 3. The ratio of the 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.

Since the expression system was different and the T7 binding site removed, it is important to keep in mind, that the original biobrick may not function like this. However, the RFP could theoretically be expressed in the fusion protein.

This data was contributed by the Aboa team 2019.

References

1. Chin, J.W. (2017) Expanding and reprogramming the genetic. Nature 550: 53-60.

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]

Sequence and Features