Part:BBa_K567018

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 .

References

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

Sequence and Features BBa_K567018 SequenceAndFeatures