Difference between revisions of "Part:BBa K1739001"
Line 2: | Line 2: | ||
<partinfo>BBa_K1739001 short</partinfo> | <partinfo>BBa_K1739001 short</partinfo> | ||
− | This part uses the BBa_J23104 and encodes | + | This part uses the BBa_J23104 and encodes Cytochrome <i>b</i><sub>562</sub> 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 <i>b</i><sub>562</sub> 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). |
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Line 18: | Line 18: | ||
===Validation=== | ===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 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. | ||
+ | |||
+ | [[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 16:08, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.