Difference between revisions of "Part:BBa K2333434"

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pLac0-1 mf-Lon

This is an IPTG-inducible mf-Lon construct containing the pLlac 0-1 promoter. It was a cornerstone in William and Mary 2017's efforts to produce a modular method to alter gene expression speed, enabling them to test a wide variety of protease concentrations with ease. All of their primary characterization was done using this part, and the construct should prove to be useful to anyone in the future who wants to test a variety of mf-Lon concentrations without having to undergo a large number of cloning steps. This part can also be used to produce fully functional circuit motifs, as was demonstrated with W&M's mScarlet IFFL cricuit, and as such could contribute to proof of concept or final implementation of other projects.

Usage and Biology

This composite part is a combination circuit with the LacI repressor under the constitutive promoter J23105 and mf-Lon under the control of the PLlac 0-1 promoter. William and Mary 2017 modified the mf-Lon gene via codon-optimization for iGEM use and added a double terminator. The mf-Lon protease specifically targets different protein degradation tags with varying affinities corresponding to varying degradation rates. This IPTG-inducible mf-Lon construct was used in tandem with aTc-inducible pdt reporter constructs by William and Mary 2017 to obtain gene expression speed measurements.

Characterization

W&M 2017 characterized this mf-Lon containing composite part in combination with aTc-inducible pdt reporter constructs as well as with copper sulfate inducible pdt reporter constructs. The graphs below show this speed data along with the data from the other tags in their series (e.g. BBa_K2333413-BBa_K2333419).

mScarlet Experiments

BBa_K2333427-BBa_K2333433

Graph 1: Time course measurements were performed according to standard protocol, and fluorescence was normalized to steady state based upon when fluorescence no longer increased. Data is shown for each construct until steady state is reached (this means at least two consecutive subsequent data points do not increase fluorescence). As the no-pdt condition had not reached steady state when time course was ended, it was normalized to the final collected data point, which is likely close to the true steady state. Geometric mean of 10,000 cells each of three biological replicates. Shaded region represents one geometric standard deviation above and below the mean.

Graph 2: Comparison of calculated t1/2 vs degradation rate. Degradation rate was obtained as above, and t1/2 was defined as time at which each biological replicate's regression line reached half of steady state. The blue line represents an optical guide for the eye, and is not fitted. Speed is scaling with degradation rate and following a predicted trend.

Graph 3: Degradation rates were measured in the above constructs. Each data point represents the population geometric mean of at least 10,000 cells of a distinct biological replicate. Relative degradation was calculated relative to the geometric mean fluorescence of the untagged control.

Graph 4: Comparison of calculated t1/2 to construct pdt. Degradation rate was obtained, and t1/2 was defined as time at which each biological replicate's regression line reached half of steady state. Each data point represents the population geometric mean of at least 10,000 cells of a distinct biological replicate.

IFFL MEFL

Graph 5: Measurements of absolute gene expression using aTC inducible mScarlet-I constructs. Data is shown for each construct until steady state is reached (this means at least two consecutive subsequent data points do not increase fluorescence). Geometric mean of 10,000 cells each of three biological replicates. Shaded region represents one geometric standard deviation above and below the mean.

Copper Experiments

BBa_K2333437-BBa_K2333442

Graph 6: Results for a plate reader functionality assay. Cells were grown for 4 hours and then induced with either 500µM or 1mM of CuSO4 (to induce copper sensing parts). After 2 hours, cells were induced further with .01mM IPTG (to induce pLac mf-Lon). Introduction of mf-Lon clearly decreased the fluorescence of the cells, and based upon the results from this assay, W&M 2017 decided to perform speed tests on these constructs with 500µM CuSO4 and .01mM IPTG. Shaded region represents one geometric std above and below mean.

W&M 2017 was able to see a significant, but somewhat noisy speed change. While it was clear that parts with increased degradation rate were reaching steady state faster, it was fairly difficult to distinguish them from one another. This was likely due to cell death and toxicity issues with higher concentrations of copper sulfate and time concerns preventing thorough more testing of parameters.

Graph 7: Results for a plate reader functionality assay. Cells were grown for 4 hours and then induced with either 500µM or 1mM of CuSO4 (to induce copper sensing parts). After 2 hours, cells were induced further with .01mM IPTG (to induce pLac mf-Lon). Introduction of mf-Lon clearly decreased the fluorescence of the cells, and based upon the results from this assay, W&M 2017 decided to perform speed tests on these constructs with 500µM CuSO4 and .01mM IPTG.

Sequence and Features

References

[1] Cameron DE, Collins JJ. Tunable protein degradation in bacteria. Nature Biotechnology. 2014;32(12):1276–1281.

[2] Torella JP, Boehm CR, Lienert F, Chen J-H, Way JC, Silver PA. Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Research. 2013;42(1):681–689.