Translational_Unit

Part:BBa_K567015

Designed by: You Wang   Group: iGEM11_SJTU-BioX-Shanghai   (2011-09-29)

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 2011 SJTU-BioX-iGEM


Related Biobrick:

PT7-metGM (BBa_K567014)

metY-CGA (BBa_K567016)

Improved Part

Improved part: BBa_K2443013 submitted by Lethbridge iGEM 2017

Rational behind improvements: 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 E. coli. 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-Gold (DE3) gold cell by putting it under the control of a T7 promoter (BBa_I719005), RBS (BBa_B0034) and double terminator (BBa_B0015). The original part is also incompatible with biobrick standards. To address this we have removed all illegal cut sites from the construct.


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]



Functional Parameters: Austin_UTexas

BBa_K567015 parameters

Burden Imposed by this Part:

Burden Value: 1.6 ± 2.2%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

[edit]
Categories
//cds/enzyme
Parameters
chassisER2566
functionanimo acylate tRNA(Met)
ligandsIsopropyl β-D-1-Thiogalactopyranoside (IPTG)