Difference between revisions of "Part:BBa K1985013"
| Line 13: | Line 13: | ||
<partinfo>BBa_K1985013 parameters</partinfo> | <partinfo>BBa_K1985013 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
| − | |||
==Validation== | ==Validation== | ||
Revision as of 22:25, 19 October 2016
Sequence coding for Sup35 with residues 1-61 with an arabinose inducible promoter
This part is an improved version of a previously designed BioBrick (Part:BBa_K1739002) from the Kent 2015 iGEM team. This part contains three segments, an arabinose inducible promoter designed by the Imperial 2014 iGEM team (Part:BBa_K1321333), the CsgA signal sequence and only the first 61 aminoacids residues of the prion domain Sup35. Our improved BioBrick aims to optimize the self-assembly process of amyloid fibrils with the addition of these residues as they have been considered to be a suitable building block for the assembly of functional nanostructures***. The addition of the arabinose inducible promoter allows for tighter control for the expression of amyloid fibres rather than a constitutive promoter as was previously used. This part was inserted into part (Part:BBa_K1985016), which is a pSB1A3 backbone with (Part:BBa_K1321333).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Validation
The improved plasmid was validated in three ways:
Restriction Digest
The plasmid was analysed through a diagnostic double restriction cut, using the enzymes EcoRI and BamHI. This was followed by agarose gel electrophoresis. The enzymes cleave the pBAD Ara-C promoter at 1165 bp, with the remainder plasmid and the insert being 2526 bp. The size of the two fragments were compared with size of the Invitrogen 1kB DNA marker and was found that our fragments were the correct.
Congo Red Plate Assay
This assay was used for validation as it confirms the presence of self assembled amyloid that our BioBrick encodes for. The Congo red plates were compared to a negative control strain, VS45 with pVS105. pVS105 contains CsgAss with Sup35M, that does not produce a protein product that self assembles into amyloid. The results (shown in Fig.2) confirmed that our protein self-assembled into amyloid nano-wires due to the red color of the colonies, in comparison to the white negative control colonies, which demonstrates no presence of amyloid nano-wires.