|
|
| Line 4: |
Line 4: |
| | The tRNA(Arg) is under the control of promoter trc. tRNA(Arg) expression is induced by 0.5mM IPTG when the OD600 of the culture reaches 0.3. | | The tRNA(Arg) is under the control of promoter trc. tRNA(Arg) expression is induced by 0.5mM IPTG when the OD600 of the culture reaches 0.3. |
| | | | |
| − | =='''Background'''== | + | ===Design=== |
| − | '''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 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002] ''sulA'' promoter-tRNA<sup>Arg</sup>-AGG) or trc promoter([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]'' lacI''-Ptrc-tRNA<sup>Arg</sup>) to control rare tRNA amount.
| + | In this part we have overexpressed rare tRNA<sup>Arg</sup>-AGG in the cell. The rare tRNA can recognize AGG codon on the mRNA. |
| | | | |
| − | '''tRNA Modulator''': | + | '''tRNA<sup>Arg</sup>-AGG'''([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]): tRNA<sup>Arg</sup>-AGG is over expressed under the control of trc promoter (induced by IPTG). |
| | | | |
| − | *''sulA'' promoter-tRNA<sup>Arg</sup>-AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002])
| + | This rare tRNA<sup>Arg</sup> can be charged with Arg by native '''Arginyl-tRNA Synthetase(ArgRS)''' in E.coli. |
| | | | |
| − | *''lacI''-Ptrc-tRNA<sup>Arg</sup>-AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001] )
| + | [[image:11SJTU_Rare_03.jpg|center|Over expressed tRNA(Arg)-AGG is charged by native Arginyl-tRNA Synthetase(ArgRS)]] |
| | | | |
| − | '''Reporter''' can control two elements: the number of rare codons inserted into luciferase after start codon ATG and the strength of target protein promoter. | + | '''RFP-6AGG'''([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567017 BBa_K567017]): we have inserted 6 AGG codons after the start codon ATG in the RFP gene. |
| − | In our project, 2, 4, 6, 8 rare codon AGGs are inserted into the Reporter gene. T7 promoter or bla promoter are used to control target protein mRNA amount.
| + | |
| | | | |
| − | '''Reporter''':
| + | [[image:11SJTU_rare_00.jpg|500px|center| 6AGG codons are inserted after the start codon ATG in the reporter gene]] |
| | | | |
| − | *P''bla''-Luc-2AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567004 BBa_K567004])
| + | ===Action=== |
| | | | |
| − | *P''bla''-Luc-4AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567005 BBa_K567005])
| + | When rare tRNA<sup>Arg</sup>-AGG is not over-expressed, RFP expression is hindered. When tRNA<sup>Arg</sup>-AGG is over-expressed, this tRNA can recognize the AGG codon on the mRNA so a large amount of RFP is produced. |
| | | | |
| − | *P''bla''-Luc-6AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567006 BBa_K567006])
| + | [[image:11SJTU_rare_01.jpg|center]] |
| | | | |
| − | *P''bla''-Luc-8AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567007 BBa_K567007])
| |
| | | | |
| − | *PT7-Luc-2AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567008 BBa_K567008])
| + | ----- |
| | | | |
| − | *PT7-Luc-4AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009])
| + | ===Result=== |
| | | | |
| − | *PT7-Luc-6AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567019 BBa_K567019])
| + | [[File:11SJTU_rare_02.png|thumb|500px|center|''Fig.1'' Confocal Microscope examining RFP expression. '''RFP has been largely produced in cells overexpressing tRNA<sup>Arg</sup>-AGG.''']] |
| | | | |
| − | *PT7-Luc-8AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010])
| + | [[File:11SJTU_rare_10.jpg|thumb|500px|center|''Fig.2'' Cells over-expressing tRNA<sup>Arg</sup>-AGG emit bright red fluorescence as wild type RFP (first one from the left). Control (first one from the right) exhibits no red fluorescence.]] |
| | | | |
| − | '''Note:'''Luc for luciferase
| + | RFP has been largely produced in cells overexpressing tRNA<sup>Arg</sup>. No RFP can be observed in cells without rare tRNA overexpression. |
| | | | |
| − | 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. | + | '''We have successfully controlled protein expression by controlling rare tRNA amount.''' |
| | | | |
| | | | |
| − | =='''The Working Curve of tRNA Modulator'''== | + | [[image:11SJTU_arrow_next.jpg|right|Next Page|link=Team:SJTU-BioX-Shanghai/Project/Subproject1-2]] |
| | | | |
| − | 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:
| |
| − |
| |
| − | [[image:11-SJTU-compare-4.jpg|frame|center|''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<sup>Arg</sup> ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter P''bla''-Luc-4AGG（[https://parts.igem.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<sup>Arg</sup> ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter PT7-Luc-4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]). The working curve of tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup> 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:
| |
| − |
| |
| − | <gallery caption="Pbla">
| |
| − | image:11SJTUResultPlbla6.jpg|''Fig.2''
| |
| − | image:11SJTUResultPbla8.jpg|''Fig.3''
| |
| − | </gallery>
| |
| − |
| |
| − | <gallery caption="PT7">
| |
| − | image:11SJTUT7-2.png|''Fig.4''
| |
| − | image:11SJTUT7-6.png|''Fig.5''
| |
| − | image:11SJTUT7-8.png|''Fig.6''
| |
| − | </gallery>
| |
| − | '''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<sup>Arg</sup> ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) compared with UV excitation induced tRNA Modulator ''sulA'' promoter-tRNA<sup>Arg</sup>([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002]) , though ''sulA'' promoter-tRNA<sup>Arg</sup> responded quicker to signals. We step further to test the influence of different Reporters with tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup>.
| |
| − |
| |
| − |
| |
| − | =='''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.
| |
| − |
| |
| − | [[image:11SJTUzhuzhuangtu-compare.jpg|frame|center|''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.
| |
| − |
| |
| − | [[image:11sjtu_Compare_8.png|frame|center|''Fig.8'' (A) Working curve of tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup> under Reporter P''bla''-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. ]]
| |
| − |
| |
| − |
| |
| − | =='''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:
| |
| − |
| |
| − | [[image:11sjtu_Compare_bla48_T72468.png|frame|center|''Fig.9'' (A) Comparing the influence of different number of rare codon insertions in tRNA Modulator working curve. Two Reporters are compared, P''bla''-Luc-4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567005 BBa_K567005]) and P''bla''-Luc-8AGG ([https://parts.igem.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([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567008 BBa_K567008]), PT7-Luc-4AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]), PT7-Luc-8AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010]) and PT7-Luc-6AGG ([https://parts.igem.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.
| |
| − |
| |
| − | [[image:11sjtu_Compare_DATA_Fitting.png|frame|center|''Fig.11'' The comparison between background expression and induced expression of luciferase with different rare codon insertions. (A)PT7-luc reporters. (B) Pbla-luc reporters]]
| |
| − |
| |
| − | 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 ([https://parts.igem.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 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]) with the same tRNA Modulator. The result is shown below:
| |
| − | [[image:11SJTUResultPT7_4_and_8(4-2).jpg|frame|center|''Fig.10'' the comparison of enzyme activity between PT7-Luc-4AG and PT7-Luc-2x4AGG under tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup>. ]]
| |
| − |
| |
| − | 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=== | | ===Reference=== |
The tRNA(Arg) is under the control of promoter trc. tRNA(Arg) expression is induced by 0.5mM IPTG when the OD600 of the culture reaches 0.3.
