Difference between revisions of "Part:BBa K1985014"

Revision as of 23:24, 19 October 2016

Sequence coding for Sup35(residues 1-61) and Cytochrome b562 with an arabinose inducible promoter

This part is an improved version of the fusion protein (Part:BBa_K1739003) designed by the Kent 2015 iGEM team. It contains four segments, an arabinose inducible promoter designed by the Imperial 2014 iGEM team (Part:BBa_K1321333), the CsgA signal sequence, the first 61 aminoacids of the prion domain Sup35 and the electron transfer protein cytochrome 562. 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).

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 2876 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.

Figure 1.1% agarose gel of the restriction digest of BBa_K1985014 in pSB1A3 plasmid backbone with EcoRI and BamHI. Single digest with EcoRI shows undigested plasmid, as two bands can be seen

Congo Red 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.

AFM Imaging

Figure 1.1% agarose gel of the restriction digest of BBa_K1985014 in pSB1A3 plasmid backbone with EcoRI and BamHI. Single digest with EcoRI shows undigested plasmid, as two bands can be seen

Figure 1.1% agarose gel of the restriction digest of BBa_K1985014 in pSB1A3 plasmid backbone with EcoRI and BamHI. Single digest with EcoRI shows undigested plasmid, as two bands can be seen

Sequence and Features