Difference between revisions of "Part:BBa K1739001"
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===Validation===
 
===Validation===
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To validate the nature of our part, we analysed it through agarose gel electrophoresis. A restriction digest preceded this, using the enzymes ECORI and PSTI. The enzymes cleave pSBIC3 into a fragment of 2030bp. By comparing the sizes of the fragments to a marker we discovered the size of our insert ( as seen in figure 1) to be around 400bp, matching the actual size of 364 base pairs of the Cyt<i>b</i><sub>562</sub> sequence. Also, our gel shows a fragment to be about 2000 base pairs, confirming it is pSB1C3 which is 2030 base pairs in size.
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To validate the nature of our part, we analysed it through agarose gel electrophoresis. A restriction digest preceded this, using the enzymes ECORI and PSTI. The enzymes cleave pSBIC3 into a fragment of 2029bp, with our insert being 345bp. By comparing the sizes of the fragments to the Invitrogen 1kB DNA Marker, we found that our fragments had separated to the expected sizes (see figure 1.), therefore validating our BioBrick.  
  
 
[[File:Team Kent Cytb562gel labelled.jpg|thumb|center|400px|Figure 1. Shows the agarose gel, following a restrictive digest of our BioBrick containing Cytochrome <i>b</i><sub>562</sub>.]]
 
[[File:Team Kent Cytb562gel labelled.jpg|thumb|center|400px|Figure 1. Shows the agarose gel, following a restrictive digest of our BioBrick containing Cytochrome <i>b</i><sub>562</sub>.]]

Revision as of 17:42, 18 September 2015

Sequence coding for Cytochrome b562

This part uses the BBa_J23104 and encodes Cytochrome b562 in a pSB1C3 backbone. This part has been validated by digestion and quantification of the presence of the cytochrome gene on a diagnostic gel. Cytochrome b562 is a single subunit, four-helix bundle protein containing a non-covalently bound b-type haem group with a molecular weight of 25kDa (Fujiwara, Fnkumori, and Yamanaka, 1993; Robinson et al., 1997).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Validation

To validate the nature of our part, we analysed it through agarose gel electrophoresis. A restriction digest preceded this, using the enzymes ECORI and PSTI. The enzymes cleave pSBIC3 into a fragment of 2029bp, with our insert being 345bp. By comparing the sizes of the fragments to the Invitrogen 1kB DNA Marker, we found that our fragments had separated to the expected sizes (see figure 1.), therefore validating our BioBrick.

Figure 1. Shows the agarose gel, following a restrictive digest of our BioBrick containing Cytochrome b562.


References:

Fujiwara, T., Fnkumori,, Y. and Yamanaka, T. (1993). Halobacterium halobium Cytochrome b-558 and Cytochrome b-562: Purification and Some Properties. J. Biochem., 113, pp.48-54.

Robinson, C., Liu, Y., Thomson, J., Sturtevant, J. and Sligar, S. (1997). Energetics of Heme Binding to Native and Denatured States of Cytochrome b 562 †. Biochemistry, 36(51), pp.16141-16146.