Difference between revisions of "Part:BBa K567012"

Latest revision as of 03:30, 29 October 2011

tRNA(Asp)-AGG

tRNA^Arg with its anticodon mutated to CCU(base pairing rare codon AGG) and under the control of aspV promoter. This biobrick is constructed first by cloning the tRNA^Arg from aspV in E.coli, then the anticodon region is site-directed mutated.

This part is worked coordinately with TDRS(BBa_K567011). TDRS is aspartyl aminoacyl tRNA synthetase without anticodon recognition domain. This modified AspRS can charge Asp to tRNA^Asp-AGG (BBa_K567012). With tRNA^Asp-AGG and TDRS, the ribosome can get through consecutive AGG codons on the mRNA.

Reporter:We test our design with two reporters.

RFP-6AGG (BBa_K567017) : RFP with 6AGG insertions

Luciferase-4AGG (BBa_K567009) : luciferase with 4AGG insertions

When the modified enzyme is produced under induction, it can charge tRNA^Asp-AGG with Arg. Then the charged tRNA can get through the rare codons on the mRNA, so that RFP or luciferase can be produced.

Fig.1 With our device (BBa_K567012 and BBa_K567011), RFP-6AGG is expressed.

Fig.2 Red fluorescence can be observed in cells with our device (the middle three). Wild type RFP (the first one from the left) exhibits bright red fluorescenceis. Control (first one from the right) exhibits no red fluoresence.

We have used PT7-RFP-6AGG (BBa_K567017) as our Reporter. We have constructed tRNA^Asp-AGG and PT7-TDRS (AspRS without anticodon recognition domain) (BBa_K567012 and BBa_K567011). tRNA^Asp-AGG, which can recognize rare codon AGG, is under constitutive promoter. With our device, RFP is successfully produced. Without our device, little RFP is observed.

Fig.3 Examination of luciferase production with and without device. ER2566 cannot produce luciferase with PT7-Luc-4AGG (BBa_K567009) only. When tRNA^Asp-AGG (BBa_K567012) and PT7-TDRS (BBa_K567011) are co-transformed into the cell, luciferase production is increased. The results proved that aaRS can regulate protein biosynthesis.

We have used PT7-Luc-4AGG (BBa_K567009) as our Reporter to test the function of PT7-TDRS (BBa_K567011) and tRNA^Asp-AGG (BBa_K567012). Results are shown above. Luciferase production has been largely increased with our device.

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PT7-TDRS (BBa_K567011)

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 281
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 281
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 281
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 281
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 281
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K567012 short</partinfo>
-tRNA(Asp) with its anticodon mutated to CCU(base pairing rare codon AGG) and under the control of ''aspV'' promoter. This biobrick is constructed first by cloning the tRNA(Asp) from ''aspV'' in E.coli, then the anticodon region is site-directed mutated.
+tRNA<sup>Arg</sup> with its anticodon mutated to CCU(base pairing rare codon AGG) and under the control of ''aspV'' promoter. This biobrick is constructed first by cloning the tRNA<sup>Arg</sup> from ''aspV'' in ''E.coli'', then the anticodon region is site-directed mutated.
 This part is worked coordinately with TDRS([https://parts.igem.org/Part:BBa_K567011 BBa_K567011]). TDRS is aspartyl aminoacyl tRNA synthetase without anticodon recognition domain. This modified AspRS can charge Asp to tRNA<sup>Asp</sup>-AGG (BBa_K567012). With tRNA<sup>Asp</sup>-AGG and TDRS, the ribosome can get through consecutive AGG codons on the mRNA.
-We have used PT7-RFP-6AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567017 BBa_K567017]) as our Reporter. We have constructed tRNA<sup>Asp</sup>-AGG and PT7-TDRS (AspRS without anticodon recognition domain, )  ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567012 BBa_K567012] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K567011 BBa_K567011])as the Modulator. tRNA<sup>Asp</sup>-AGG, which can recognize rare codon AGG, is under constitutive promoter. Our device works as follows: Without Modulator, RFP with 6 consecutive AGG insertions can hardly be expressed. When Modulator works, tRNA<sup>Asp</sup>-AGG can be charged with Asp by TDRS. tRNA<sup>Asp</sup>-AGG recognizes rare codon AGG on the Reporter RFP-6AGG. The ribosome gets through this part of the mRNA more easily. RFP is expressed. Red fluorescence is observed. Our experiment results are shown below.
+'''Reporter''':We test our design with two reporters.
-[[image:11SJTU-ZBresult1.jpg|600px|frame|center|''Fig 2.'' With our Modulator ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567012 BBa_K567012]  and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K567011 BBa_K567011]), Reporter RFP-6AGG is expressed, demonstrating that our system works well. ]]
+RFP-6AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567017 BBa_K567017]) : RFP with 6AGG insertions
-[[image:11SJTU-ZBresult2.JPG|600px|frame|center|''Fig 3.'' aaRS Modulator + Reporter for Qualitative Analysis. aaRS Modulator + RFP-6AGG''(the middle three)'' emit red fluorescence. Wild type RFP ''(the first one from the left)'' exhibits bright red fluorescenceis. Control ''(first one from the right)'' exhibits no red fluoresence.]]
+Luciferase-4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]) : luciferase with 4AGG insertions
-Related Biobrick:
+When the modified enzyme is produced under induction, it can charge tRNA<sup>Asp</sup>-AGG with Arg. Then the charged tRNA can get through the rare codons on the mRNA, so that RFP or luciferase can be produced.
-tRNA<sup>Asp</sup>-AGG  ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567012 BBa_K567012])
+[[image:11SJTU_rare_02.jpg|thumb|500px|center|''Fig.1'' With our device ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567012 BBa_K567012] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K567011 BBa_K567011]), RFP-6AGG is expressed.]]
+[[image:11SJTU-ZBresult2.JPG|thumb|500px|center|''Fig.2'' Red fluorescence can be observed in cells with our device ''(the middle three)''. Wild type RFP ''(the first one from the left)'' exhibits bright red fluorescenceis. Control ''(first one from the right)'' exhibits no red fluoresence.]]
+We have used PT7-RFP-6AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567017 BBa_K567017]) as our Reporter.  We have constructed tRNA<sup>Asp</sup>-AGG and PT7-TDRS (AspRS without anticodon recognition domain) ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567012 BBa_K567012] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K567011 BBa_K567011]). tRNA<sup>Asp</sup>-AGG, which can recognize rare codon AGG, is under constitutive promoter. '''With our device, RFP is successfully produced. Without our device, little RFP is observed. '''
+[[image:11SJTU_rare_08.jpg|thumb|500px|center|''Fig.3'' Examination of luciferase production with and without device. ER2566 cannot produce luciferase with PT7-Luc-4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]) only. When tRNA<sup>Asp</sup>-AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567012 BBa_K567012]) and PT7-TDRS ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567011 BBa_K567011]) are co-transformed into the cell, luciferase production is increased. The results proved that aaRS can regulate protein biosynthesis. ]]
+We have used PT7-Luc-4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]) as our Reporter to test the function of PT7-TDRS ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567011 BBa_K567011]) and tRNA<sup>Asp</sup>-AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567012 BBa_K567012]). Results are shown above. '''Luciferase production has been largely increased with our device.'''
-tRNA<sup>Asp</sup>-TAG  ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567013 BBa_K567013])
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