From e53281691116d73b7ca93c94897691be291a661b Mon Sep 17 00:00:00 2001 From: Maarten van den Ancker <s2037423@ed.ac.uk> Date: Thu, 13 Oct 2022 18:00:09 +0000 Subject: [PATCH] Replace results.html --- wiki/pages/results.html | 10 +++++----- 1 file changed, 5 insertions(+), 5 deletions(-) diff --git a/wiki/pages/results.html b/wiki/pages/results.html index f2df982..da46db4 100644 --- a/wiki/pages/results.html +++ b/wiki/pages/results.html @@ -176,12 +176,12 @@ <p>After we had completed our level one assemblies for the mutated MerR (m_MerR), PbrR and ArsR transcription factor level one plasmids we ran a digestion on the plasmids using EcoR1 (Figure 2). The mutated MerR and the ArsR plasmids were around 3000bp, suggesting that the acceptor plasmid was ligated without the transcription factor insert. Only the PbrR was assembled correctly as we see a band around 3500bp which is the size band we expect if there is an insert in the plasmid. We then performed the m_MerR and ArsR assemblies again along with the MerR. We were unable to do this assembly along with the initial ones we did as we did not have the parts needed to make this assembly. We then ran a colony PCR on these three assemblies to check for the presence of our insert in the plasmids. In figure 3a we can see a smear at around 400bp which suggests that the level 1 JUMP plasmid contains the MerR insert. However, for the ArsR and mutated MerR, we saw no band (Figure 3b) showing that there was no insert present in the DNA and that the assembly failed. Due to time constraints, we could no longer pursue the ArsR and mutated MerR and try and get a working assembly. So, we only used the MerR and PbrR assemblies for protein expression analysis.</p> <h5>Protein Expression Analysis</h5> - <p>To perform the protein expression analysis we transformed <i>E. coli</i> Bl21(DE3) with the level 1 JUMP containing the PbrR and MerR expression cassettes and performed SDS-PAGE analysis (Figure 4). The SDS-PAGE indicated that both expression cassettes produced their respective transcription factors. The presence of the band at 15 kDa in the PbrR lane indicates that it expresses PbrR as this is its predicted size. We also saw a band at 16 kDa in the MerR lane which indicates that it expresses MerR as this is its predicted size.</p> - <figure> - <img src="https://static.igem.wiki/teams/4390/wiki/results/figure-4-biosesnors.png" style="width:65%;display:block;left:0;right:0;margin:auto;"> - <figcaption style="width:65%;left:0;right:0;margin:auto;"><b>Figure 4.</b> SDS-PAGE of the lysates of <i>E. coli</i> BL21(DE3) which were transformed with the level 1 JUMP plasmids containing the PbrR and MerR expression cassettes. Loading was standardised using a Bradford assay, with 3 mg of proteins loaded into each well. Arrows show the band which indicates we have expression of our transcription factors.</figcaption> - </figure> + <p>To perform the protein expression analysis we transformed <i>E. coli</i> Bl21(DE3) with the level 1 JUMP containing the PbrR and MerR expression cassettes and performed SDS-PAGE analysis (Figure 4). The SDS-PAGE indicated that both expression cassettes produced their respective transcription factors. The presence of the band at 15 kDa in the PbrR lane indicates that it expresses PbrR as this is its predicted size. We also saw a band at 16 kDa in the MerR lane which indicates that it expresses MerR as this is its predicted size.</p> + <figure> + <img src="https://static.igem.wiki/teams/4390/wiki/diagrams/image-20221013-163208-e46ddb24.png" style="width:20%;display:block;left:0;right:0;margin:auto;"> + <figcaption style="width:65%;left:0;right:0;margin:auto;"><b>Figure 4.</b> SDS-PAGE of the lysates of <i>E. coli</i> BL21(DE3) which were transformed with the level 1 JUMP plasmids containing the PbrR and MerR expression cassettes. Loading was standardised using a Bradford assay, with 3 mg of proteins loaded into each well. Arrows show the band which indicates we have expression of our transcription factors.</figcaption> + </figure> <h3 id="1.3" class="anchor">1.3) <i>In vitro</i> transcription</h3> <p>Once we had our linear construct and cells which expressed the transcription factors we then wanted to characterise our biosensors. To do this we performed our in-vitro transcription reactions (More information on the experiments page). We did not include the cell lysates containing the transcription factors as we wanted to see if the linear constructs would produce fluorescence without the transcriptional repression caused by the transcription factors (More information on the design page). However, none of our linear biosensors produced any fluorescence (Figure 5a) when compared to our positive control of GFP seen at the top of figure 5a. This would suggest either that there is no RNA transcription happening or that the RNA aptamer is unable to bind to the DFHBI fluorophore as it is no longer functional.</p> -- GitLab