Difference between revisions of "Part:BBa K1739000"
Line 19: Line 19:
  
 
===Validation===
 
===Validation===
−
To validate the nature of our plasmid, we analysed it through agarose gel electrophoresis. By comparing the sizes of the fragments to a marker we discovered the size of our fragment (as seen in figure 1) to be slightly less than 850 base pairs, matching the actual size of 800 base pairs of the Sup35NM sequence. Also, our gel shows a fragment to be about 2000 base pairs, confirming it is PSB1c3 which is 2030 base pairs in size.
+
To validate the nature of our plasmid, we analysed it through agarose gel electrophoresis. A restriction digest of our plasmid was carried out using ECORI and PSTI. These enzymes cleave pSBIC3 at 2029bp, with our insert being 780bp. By comparing the sizes of our inserts to the Invitrogen 1kB DNA Marker, we found that our fragments were the correct sizes, therefore validating our BioBrick.  
  
 
[[File:Team KentSUP35gel2.jpg|thumb|center|400px|Figure 1. Shows the agarose gel produced, following the restriction digest of our part.]]  
 
[[File:Team KentSUP35gel2.jpg|thumb|center|400px|Figure 1. Shows the agarose gel produced, following the restriction digest of our part.]]  

Revision as of 17:39, 18 September 2015

Sequence coding for amyloid Sup35NM

We have improved a previously designed BioBrick (Part:BBa_K401001) from the [http://2010.igem.org/Team:Valencia Valencia 2010 iGEM team] that encoded the Sup35 protein from Saccharomyces cerevisiae. The previously designed BioBrick contained two illegal cut sites for Pstl and one for Bsal within the coding region that reduce compatibility for digestion and modification of the part. Our improved BioBrick has used genome optimisation in order to remove these cut sites, producing a part compatible with the iGEM part submission standards. Validation of this part used a diagnostic Congo Red plate that demonstrated the presence of amyloid by formation of red colonies.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 522


Validation

To validate the nature of our plasmid, we analysed it through agarose gel electrophoresis. A restriction digest of our plasmid was carried out using ECORI and PSTI. These enzymes cleave pSBIC3 at 2029bp, with our insert being 780bp. By comparing the sizes of our inserts to the Invitrogen 1kB DNA Marker, we found that our fragments were the correct sizes, therefore validating our BioBrick.

Figure 1. Shows the agarose gel produced, following the restriction digest of our part.