From 688cbb77ed6b6dbba8aa955aad3e6b82f4c0b007 Mon Sep 17 00:00:00 2001
From: Arya Ajit Gohad <aryagohad21@gmail.com>
Date: Fri, 16 Dec 2022 17:12:16 +0000
Subject: [PATCH] Update wiki/pages/engineering-learn.html,
wiki/pages/engineering-design2.html
---
wiki/pages/engineering-design2.html | 13 ++++++-------
wiki/pages/engineering-learn.html | 2 +-
2 files changed, 7 insertions(+), 8 deletions(-)
diff --git a/wiki/pages/engineering-design2.html b/wiki/pages/engineering-design2.html
index c3ef17d..9876d9c 100644
--- a/wiki/pages/engineering-design2.html
+++ b/wiki/pages/engineering-design2.html
@@ -175,11 +175,9 @@ We have also done project <a href="{{ url_for('pages', page='model') }}">Modelli
</p>
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- </p><br>
- <p> We tested these sequences out in two strains of E. coli: Dh5alpha, a cloning strain and BL21(DE3), an expression strain. The analysis suggests that there is not much of a variation in the F/OD values. This suggests that probably in some cases, changing just one nucleotide does not change the expression. This may have structural correlations. <br></p>
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+ <p> We tested these sequences out in two strains of E. coli: Dh5alpha, a cloning strain and BL21(DE3), an expression strain. The analysis suggests that there is not much of a variation in the F/OD values. This suggests that probably in some cases, changing just one nucleotide does not change the expression. This may have structural correlations.</p>
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<p><h3>Characterization of CspF Mutants in DH5alpha Strain:</h3</p>
<center><iframe src="https://static.igem.wiki/teams/4303/wiki/data-for-the-sdm-utrs-in-dh5alpha.pdf" width="100%" height="500px"></iframe></center> </p>
@@ -195,16 +193,17 @@ We have also done project <a href="{{ url_for('pages', page='model') }}">Modelli
<br>
<p><h3>NUPACK CspF Mutants</h3</p>
<center><iframe src="https://static.igem.wiki/teams/4303/wiki/nupack-cspf-mutants.pdf" width="100%" height="500px"></iframe></center>
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<p> We tested these sequences out in two strains of E. coli: Dh5alpha, a cloning strain and BL21(DE3), an expression strain. The analysis suggests that there is not much of a variation in the F/OD values. This suggests that probably in some cases, changing just one nucleotide does not change the expression. This may have structural correlations. <br></p>
<p> We have correlated it with NuPACK. However, while calculating the relative performance with respect to cspF, we realised that if a G/C in the original sequence GGAATTTTT was changed to an A/T then the F/OD values went lower. Whereas, if the vice versa was done, the F/OD value increased. We already have an experimentally validated library that gives a range of expression with respect to just the RBS (negative control). Our further designs will be based in learnings and developments from this experiment. <br></p>
+ <br>
<p><h3>NUPACK ANALYSIS</h3></p>
<p><ul><li>From this, the least performing UTR SDM23 clearly has a very different structure. SDM10 has a similar structure but an Relative Performance 0.21 times higher than SDM23. This could be probably because of the GC content of it being different?</li><li>
SDM27 has the best expression and Relative Performance of 1.3. It’s structure is very different from the other UTRs. We need to perform 3D corrations.</li><li>
SDM26 and 9 have similar structures and similar Relative Performances.</li><li>
SDM3 and SDM5 have a similar structure and a similar Relative Performance.</li></ul>
</p>
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<p>We wish to modify the GC content of cspF by making multiple mutations in one sequence while maintaining its structural scaffold in the third engineering cycle. The next aspect that we wish to check is that keeping the structural scaffold of the cspF intact we wish to add additional structural complexities of different levels to it through our sequence and see how the gene expression changes.<br></p>
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diff --git a/wiki/pages/engineering-learn.html b/wiki/pages/engineering-learn.html
index 0ca45d8..3a6af34 100644
--- a/wiki/pages/engineering-learn.html
+++ b/wiki/pages/engineering-learn.html
@@ -16,7 +16,7 @@ Engineering Cycle - Learn
<!-- <center><h2>Learn</h2></center> -->
<!-- <hr> -->
<p>
- After testing out all 16 sequences in two batches of 8 and 11 each, we had all the data awaiting analysis. As a first step, we ranked all the sequences in decreasing order of their average fluorescence per optical density (F/OD) values at 24 hours mark (late-stationary phase). We tried to see if this ranking had any correlation with the expression values suggested by Salis Lab’s RBS Calculator (v.2.1.1). To our surprise, the Spearman's rank correlation coefficient came out to be 0.77 for the first batch of 6 UTRs, 0.39 for the second batch of 10 UTRs and barely 0.15 for all sequences put together. This could have happened because the RBS calculator has been designed for readings taken in the log phase. This concern was experienced by Xiao et al. in 2020, and the researchers gave a similar explanation as reasoning.
+ After testing out all 19 sequences in two batches of 8 and 11 each, we had all the data awaiting analysis. As a first step, we ranked all the sequences in decreasing order of their average fluorescence per optical density (F/OD) values at 24 hours mark (late-stationary phase). We tried to see if this ranking had any correlation with the expression values suggested by Salis Lab’s RBS Calculator (v.2.1.1). To our surprise, the Spearman's rank correlation coefficient came out to be 0.77 for the first batch of 8 UTRs, 0.39 for the second batch of 10 UTRs and barely 0.15 for all sequences put together. This could have happened because the RBS calculator has been designed for readings taken in the log phase. This concern was experienced by Xiao et al. in 2020, and the researchers gave a similar explanation as reasoning.
</p><p>
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GitLab