Team:SJTU-BioX-Shanghai/Project/Subproject1/Results 2

Rare-Codon Switch

tRNA Modulator + Reporter for Quantitative Analysis

tRNA Modulator and Reporter for Quantitative Analysis work together to regulate protein biosynthesis.

tRNA Modulator controls rare tRNA amount. In our project, we use sulA promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002] sulA promoter-tRNA^Arg-AGG) or trc promoter([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001] lacI-Ptrc-tRNA^Arg) to control rare tRNA amount.

tRNA Modulator:

• sulA promoter-tRNA^Arg-AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002])

• lacI-Ptrc-tRNA^Arg-AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001] )

Reporter can control two elements: the number of rare codons inserted into luciferase after start codon ATG and the strength of target protein promoter. In our project, 2, 4, 6, 8 rare codon AGGs are inserted into the Reporter gene. T7 promoter or bla promoter[1] are used to control target protein mRNA amount.

Reporter:

• Pbla-Luc-2AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567004 BBa_K567004])

• Pbla-Luc-4AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567005 BBa_K567005])

• Pbla-Luc-6AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567006 BBa_K567006])

• Pbla-Luc-8AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567007 BBa_K567007])

• PT7-Luc-2AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567008 BBa_K567008])

• PT7-Luc-4AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009])

• PT7-Luc-6AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567019 BBa_K567019])

• PT7-Luc-8AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010])

Note:Luc for luciferase

We have tested different combination of tRNA Modulators and Reporters and analyzed the influence of promoter strength for rare tRNA, number of rare codons in target protein mRNA and promoter strength for target protein gene respectively.

1.The Working Curve of tRNA Modulator

We have tested luciferin reaction in cells with different combinations of tRNA Modulators and Reporters. We examined the changes in luciferase enzyme activity over time after rare tRNA expression is induced. The amount of luciferase is reflected indirectly by the bioluminescence emitted from the luciferin reaction. Results are shown below:

Fig.1 (A)Enzyme activity of luciferase shown by bioluminescence emitted from the luciferin reaction reflecting the working curve of tRNA Modulator lacI-Ptrc-tRNA^Arg ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter Pbla-Luc-4AGG（[http://partsregistry.org/wiki/index.php?title=Part:BBa_K567006 BBa_K567006]). (B)Enzyme activity of luciferase shown by bioluminescence emitted from the luciferin reaction reflecting the working curve of tRNA Modulator lacI-Ptrc-tRNA^Arg ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter PT7-Luc-4AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]). The working curve of tRNA Modulator lacI-Ptrc-tRNA^Arg fits typical titration curve.

Here we use the above two curves as examples to characterize the working curve of tRNA Modulator. Both curves fits typical titration curve, indicating that tRNA Modulator can function as a regulating tool.

The rest of the working curves are shown here:

Pbla

Fig.2

Fig.3

PT7

Fig.4

Fig.5

Fig.6

Note:Click to see large figures.

From this experiment, we noticed that the typical working curve of tRNA Modulator can be better observed with IPTG induced tRNA Modulator lacI-Ptrc-tRNA^Arg ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) compared with UV excitation induced tRNA Modulator sulA promoter-tRNA^Arg([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002]) , though sulA promoter-tRNA^Arg responded quicker to signals. We step further to test the influence of different Reporters with tRNA Modulator lacI-Ptrc-tRNA^Arg.

2.Target Protein mRNA Amount: regulated by different strength of target protein promoters, T7 promoter and bla promoter

We examined the influence of different Reporter promoters on the working curve of tRNA Modulator, which is reflected by luciferase activity. Results showed that all the tRNA Modulator working curves fit titration curve, indicating that tRNA Modulator can act as a satisfying regulating tool.

Fig.7 Enzyme activity of luciferase reaching plateau phase.

However, the working range of tRNA Modulator is pre-defined by the strength of target protein promoter, T7 promoter and bla promoter in our project.

Fig.8 (A) Working curve of tRNA Modulator lacI-Ptrc-tRNA^Arg under Reporter Pbla-Luc-8AGG reflected by bioluminescence emitted from the luciferin reaction. (B) Working curve of tRNA Modulator under Reporter PT7-Luc-8AGG. Here we analyze the influences of strong/weak promoter in the working curve of tRNA Modulator. Moreover, strong promoter (T7) of target gene can improve the titration curve of tRNA Modulator, indicating that tRNA Modulator works better under strong target protein promoters.

3.Influence of different numbers of AGG codons put after start codon ATG in tRNA Modulator Working Curve.

The working curve of tRNA Modulator still fit titration curve under Reporters with different number of AGG insertions, indicating that the number of rare codons in the Reporter will not affect the stability and function of the Modulator. The influence of different number of rare codons in regulating protein biosynthesis is shown below:

Fig.9 (A) Comparing the influence of different number of rare codon insertions in tRNA Modulator working curve. Two Reporters are compared, Pbla-Luc-4AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567005 BBa_K567005]) and Pbla-Luc-8AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567007 BBa_K567007]). (B) Comparing the influence of different number of rare codon insertions in tRNA Modulator working curve. Four Reporters are examined, including PT7-Luc-2AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567008 BBa_K567008]), PT7-Luc-4AGG([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]), PT7-Luc-8AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010]) and PT7-Luc-6AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567019 BBa_K567019]).

Results show that the more rare codons are inserted, the lower the background expression and the narrower the range of tRNA Modulator can regulate. Besides, since the number of rare codons will not affect system stability, it can be an excellent independent regulating factor.

This picture reflects more clearly that the more rare codons are inserted, the lower the background expression and the narrower the range of tRNA Modulator regulation. We are able to predict the outcome of influence of different number of rare codons in protein biosynthesis, offering valuable information for device usage.

Future work:

We have obtained PT7-Luc-2x4AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567021 BBa_K567021]) unexpectedly during our experiment. We have tested the enzyme activity of this part under tRNA Modulator control and have compared the curve with PT7-Luc-4AGG ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]) with the same tRNA Modulator. The result is shown below:

Fig.10 the comparison of enzyme activity between PT7-Luc-4AG and PT7-Luc-2x4AGG under tRNA Modulator lacI-Ptrc-tRNA^Arg.

The result reflects the outstanding performance of the newly gained part. Two 4AGGs occurred in the beginning of the gene can bring lower background expression and much higher induced expression compared with luciferase with singe 4AGG insertion. The additional 4AGG does not affect the titration feature of the curve.

Though we may not able to explain this phenomenon now, we have realized that exploring the potential regulating modes in our system is promising and significant. We will spare no effort in exploring and perfecting our system in our future work.

Reference

[1]Ulrich Deuschlel., et al., Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures The EMBO Journal vol.5 no. 11 pp.2987-2994, 1986