Part:BBa_K1739001

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 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 Cytb₅₆₂ sequence. Also, our gel shows a fragment to be about 2000 base pairs, confirming it is pSB1C3 which is 2030 base pairs in size.

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.