Difference between revisions of "Part:BBa K1648004"
 
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[[File:K1648004.png|350px|thumb|right|Construct Map for K1648004]]
 
[[File:K1648004.png|350px|thumb|right|Construct Map for K1648004]]
  
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Consist of six fragments from magnetosome island of ''Magnetospirillum gryphiswaldense'' genome.
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Consist of a gene (mamC) from magnetosome island of ''Magnetospirillum gryphiswaldense'' genome, a promotor & RBS([https://parts.igem.org/Part:BBa_J13002 J13002]) in front of multiple restriction sites and a double terminator([https://parts.igem.org/Part:BBa_B0015 B0015]) at the back of multiple restriction sites.
  
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Due to potential difficulty in introducing the large magnetosome formation operons into ''Azotobacter vinelandii'', a "template" consists of flanking sequences from '''mamXY, mamGFDC and mms6 operons''' except the mamC operon is designed for '''homologous recombination'''.  
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Different genes can be added into this part at multiple restriction sites, so that different proteins will be expressed on the magnetosome after transformation of the part into <i>A. vinelandii</i> genome via <b>random integration</b>.
  
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Part containing this template ([https://parts.igem.org/Part:BBa_K1648002 K1648002]) together with another part containing mamC ([https://parts.igem.org/Part:BBa_K1648XXX K1648XXX]) is introduced into ''A. vinelandii'' genome via '''random integration''' before full operon sequences to be transformed into ''A. vinelandii''.
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A GFP-nanobody([https://parts.igem.org/Part:BBa_K1648005 K1648005]) is added to this part in order to express GFP-nanobody on the magnetosome. This new part is [https://parts.igem.org/Part:BBa_K1648006 K1648006]
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This Part were verified by double digestion (Fig. 1) and sequencing.
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[[File:K1648004gel.jpg]]
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Fig 1. The photo of 1% agarose gel electrophoresis showing PCR products. (A) L: DNA ladder. Lane 1: PCR products of linear Insertion Kit.
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RFP is inserted in Insertion Kit for biobrick standard. The Insertion kit with RFP is transformed in E. coli (DH5 alpha).
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Expression of RFP in Insertion is shown(Fig 2.).
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[[File:20150919_021936.jpg|350px]]
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Fig 2. Insertion kit with RFP(BBa_K1648004) is expressed in E. coli (DH5 alpha)
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 13:58, 25 September 2015

Insertion Kit for Fusing Protein of Interest to Magnetosome Membrane

Construct Map for K1648004

Consist of a gene (mamC) from magnetosome island of Magnetospirillum gryphiswaldense genome, a promotor & RBS(J13002) in front of multiple restriction sites and a double terminator(B0015) at the back of multiple restriction sites.

Different genes can be added into this part at multiple restriction sites, so that different proteins will be expressed on the magnetosome after transformation of the part into A. vinelandii genome via random integration.

A GFP-nanobody(K1648005) is added to this part in order to express GFP-nanobody on the magnetosome. This new part is K1648006

This Part were verified by double digestion (Fig. 1) and sequencing.

K1648004gel.jpg

Fig 1. The photo of 1% agarose gel electrophoresis showing PCR products. (A) L: DNA ladder. Lane 1: PCR products of linear Insertion Kit.


RFP is inserted in Insertion Kit for biobrick standard. The Insertion kit with RFP is transformed in E. coli (DH5 alpha). Expression of RFP in Insertion is shown(Fig 2.).

20150919 021936.jpg

Fig 2. Insertion kit with RFP(BBa_K1648004) is expressed in E. coli (DH5 alpha)

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2759
    Illegal XbaI site found at 2765
    Illegal SpeI site found at 1
    Illegal PstI site found at 7
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2759
    Illegal SpeI site found at 1
    Illegal PstI site found at 7
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2759
    Illegal XhoI site found at 1162
    Illegal XhoI site found at 2188
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2759
    Illegal XbaI site found at 2765
    Illegal SpeI site found at 1
    Illegal PstI site found at 7
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2759
    Illegal XbaI site found at 2765
    Illegal SpeI site found at 1
    Illegal PstI site found at 7
    Illegal NgoMIV site found at 2546
    Illegal NgoMIV site found at 2636
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 2611