Difference between revisions of "Part:BBa K1722003"
 
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{| style="color:black" cellpadding="5" cellspacing="1" border="3" align="right"
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<partinfo>BBa_K1722003 short</partinfo>
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! colspan="2" style="background:#d7474e;"|AckRS
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|-
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|'''Function'''
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|tRNA synthetase
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|-
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|'''Use in'''
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|E.coli&Mammalian cells
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|-
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|'''RFC standard'''
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|[https://parts.igem.org/Help:Assembly_standard_10 RFC 10]
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|-
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|'''Backbone'''
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|[https://parts.igem.org/Help:Plasmid_Backbones pSB1C3]
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|-
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|'''Submitted by'''
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|[http://2015.igem.org/Team:SZU_China SZU_China 2015]
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=====<partinfo>BBa_K1722003 short</partinfo>=====
  
 
AckRS is a gene which can produce a kind of aminoacyl tRNA synthetase(aaRS) that can achieve the attachment of Acetyllysine(Ack) and tRNA.Ack is a kind of unnatural amino acid(UAA) that is close structural analog of Lysine, a canonocal amino acid.
 
AckRS is a gene which can produce a kind of aminoacyl tRNA synthetase(aaRS) that can achieve the attachment of Acetyllysine(Ack) and tRNA.Ack is a kind of unnatural amino acid(UAA) that is close structural analog of Lysine, a canonocal amino acid.
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In the last decade, methods for the translational incorporation of UAAs using orthogonal aaRS-tRNA(CUA) pairs were developed. With modified aaRS which can specifically recognise a type of UAA, the UAA can be site-specifically incorporated to produce a protein with new structure and function or to expand the genetic code. The wild-type pyrrolysyl-tRNA synthatase(PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of UAAs<sup>[3]</sup> and the AckRS that we used in our project is one of them.
 
In the last decade, methods for the translational incorporation of UAAs using orthogonal aaRS-tRNA(CUA) pairs were developed. With modified aaRS which can specifically recognise a type of UAA, the UAA can be site-specifically incorporated to produce a protein with new structure and function or to expand the genetic code. The wild-type pyrrolysyl-tRNA synthatase(PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of UAAs<sup>[3]</sup> and the AckRS that we used in our project is one of them.
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AckRS is 1260bp in length. <b>Fig. 2</b> shows the DNA sequence of AckRS is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of hUPll PCR product is rather high compared with DNA Marker, which indicates that the PCR product of AckRS is in a high concerntration.
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<figure style="text-align: center"><img style="width:30%" src="https://static.igem.org/mediawiki/2015/d/d9/AckRS_pcr1.png"/><figcaption style="text-align:center"><b>Figure 2.</b> Electrophoretic analysis of PCR produution of AckRS from psi-Check2. <figcaption style="text-align:center">(1:DL2000 DNA Marker 2:PCR production 3:DL15000 DNA Marker)</figcaption></figure>
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===Design Notes===
 
===Design Notes===

Latest revision as of 02:22, 8 September 2015

AckRS
Function tRNA synthetase
Use in E.coli&Mammalian cells
RFC standard RFC 10
Backbone pSB1C3
Submitted by [http://2015.igem.org/Team:SZU_China SZU_China 2015]
AckRS can produce a aminoacyl tRNA synthetase.

AckRS is a gene which can produce a kind of aminoacyl tRNA synthetase(aaRS) that can achieve the attachment of Acetyllysine(Ack) and tRNA.Ack is a kind of unnatural amino acid(UAA) that is close structural analog of Lysine, a canonocal amino acid.

In the orthogonal system of the project of 2015 SZU-iGEM, the construction of our UAA orthogonal system rely on an orthogonal pair of tRNA(CUA) and an AckRS charging the tRNA with Ack. tRNA(CUA) has an anticodon CUA, which can pair with UAG, the amber mutated stop codon, perfectly. With the help of AckRS, the unnatural amino acid Ack can be incorporated into proteins.

aaRS functioning in the form of polycomplex in living cells.[1,2] Research on Structural Biology and Bioinformatics shows that aaRS can combine with other proteins, forming highly organized complex, to be involved in many vital physiological processes.[1,2] The structure of aaRS cantain Catalytic Central Domain(CCD) and Anticodon Binding Domain(ABD).(Fig.1)

Figure 1. The structure of aaRS(LysRS-1)
CCD:Catalytic Central Domain ABD:Anticodon Binding Domain

In the last decade, methods for the translational incorporation of UAAs using orthogonal aaRS-tRNA(CUA) pairs were developed. With modified aaRS which can specifically recognise a type of UAA, the UAA can be site-specifically incorporated to produce a protein with new structure and function or to expand the genetic code. The wild-type pyrrolysyl-tRNA synthatase(PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of UAAs[3] and the AckRS that we used in our project is one of them.

AckRS is 1260bp in length. Fig. 2 shows the DNA sequence of AckRS is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of hUPll PCR product is rather high compared with DNA Marker, which indicates that the PCR product of AckRS is in a high concerntration.

Figure 2. Electrophoretic analysis of PCR produution of AckRS from psi-Check2.
(1:DL2000 DNA Marker 2:PCR production 3:DL15000 DNA Marker)

Design Notes

The AckRS that we achieved from Shenzhen Second People's Hospital was carried by psi-Check2, which was Amp resistence. After sequencing, we found AckRS has two EcoR1 enzyme cutting site, which are the same as the site in the flank of pSB1C3. So we mutate one of the base pairs on each of the two sites. The plasmid with AckRS that we submitted is EcoR1 free.

Source

We achieved this part from Shenzhen Second People's Hospital, which is Shenzhen University First Hospital. They were doing reseach on unnatural amino acid orthogonal system and fortunately, they agree to provide us the gene.

References

[1] Hausmann C D, Ibba M. Aminoacyl- tRNA synthetase complexes: molecular multitasking revealed. FEMS Microbiol Rev, 2008, 32(4): 705 ~ 721

[2] Han JM, Kim J Y, Kim S. Molecular network and functional implications of macromolecular tRNA synthetase complex. Biochem Biophys Res Commun, 2003, 303(4): 985~ 993

[3] Veronika F, Milan V, Sabine S. Structural basis for the site-specific Incorporation of Lysine Derivatives into Proteins. Plos One, 2014, 9(4): 1~7


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1261
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 450
  • 1000
    COMPATIBLE WITH RFC[1000]