Difference between revisions of "Part:BBa K567015"
Line 27: Line 27:
  
 
''metY''-CGA  ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567016 BBa_K567016])
 
''metY''-CGA  ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567016 BBa_K567016])
−
<h1> Improved part </h1>
+
−
<p class="text12"> <b> Improved part:</b> BBa_K2443013
+
−
                <br><b>Submitted by:</b> Lethbridge iGEM 2017
+
−
                <br><b>Designed by:</b> Lethbridge iGEM 2017
+
−
                <br><b>Rational behind improvements:</b>
+
−
                BBa_K567015 encodes for a truncated MetRS without the anticodon recognition domain. We have improved this part by including the entire coding sequence, which has been codon optimized for optimal expression in <i>E. coli</i>. For easy purification we have included a C- terminal hexahistidine tag with a serine glycine linker. We have also optimized MetRS to be overexpressed in BL21 DE3 gold cell by putting it under the control of a T7 promoter (<a
+
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 21:47, 30 October 2017

PT7-metGN

T7 promoter-metG(truncated). This biobrick is constructed by putting the truncated metG (MetRS) under the control of T7 promoter and lac operator. We have cloned metG from E.coli and the tRNA recognition domain of MetRS is truncated. KanR gene with start codon substituted for CGA is used to testify the function of mutated metG. When this biobrick and metY-CGA (BBa_K567016) are co-transformed into the cell, the cells can survive on the LB Kana plate.

Construction of BBa_K567015

Rational design of MetRS: we have obtained a truncated MetRS without anticodon recognition domain. The structure of the truncated protein is shown below. This modified MetRS can charge tRNAMet without recognizing its anticodon.

Fig. This design is based on the crystal structure of methionyl-tRNA synthetase complex with tRNA (PDB ID：2CSX). We have superimposed the crystal structure of methionyl-tRNA synthetase from E.coli and obtained the overlay structure after kinetics optimization. Above is the picture showing E.coli methionyl-tRNA synthetase with (left) and without (right) anticodon recognition domain. The picture proposed that E.coli methionyl-tRNA synthetase will lose the ability to bind tRNAMet anticodon if anticodon recognition domain is deleted, thus losing anticodon specificity while maintaining aminoacylation ability. We have built the truncated MetRS, PT7-metGN (BBa_K567015), based on this design.


Characterization of BBa_K567015

When this part, metY-CGA (BBa_K567016) and KanR (BBa_K567020) are co-transformed into the cell, the cell is expected to survive Kanamycin. We tested the activity of the truncated MetRS PT7-metGN (BBa_K567015)and found that the truncated MetRS acted as expected, losing specificity for tRNAMet anticodon while maintaining aminoacylation ability.

Fig. Growth of ER2566 with a. metGN + metY-CGA, b. metGM + metY-CGA, c. + metGN, d. + metGM. Growth medium (left): LB Kana+Tet. Growth medium (right): LB Kana.

Cell growth shows that the cells show Kanamycin resistance only when both modified MetRS (metGN) and modified tRNAMet(metY-CGA) are transformed into the cell, proving that tRNA metY-CGA can transfer fMet to CGA when it is used as the start codon

For more information concerning this part, please see [http://2011.igem.org/Team:SJTU-BioX-Shanghai 2011 SJTU-BioX-iGEM]


Related Biobrick:

PT7-metGM (BBa_K567014)

metY-CGA (BBa_K567016)

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1265
    Illegal EcoRI site found at 1437
    Illegal XbaI site found at 48
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1265
    Illegal EcoRI site found at 1437
    Illegal NheI site found at 1226
    Illegal NotI site found at 1290
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1265
    Illegal EcoRI site found at 1437
    Illegal BamHI site found at 1259
    Illegal XhoI site found at 1211
    Illegal XhoI site found at 1299
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1265
    Illegal EcoRI site found at 1437
    Illegal XbaI site found at 48
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1265
    Illegal EcoRI site found at 1437
    Illegal XbaI site found at 48
  • 1000
    COMPATIBLE WITH RFC[1000]