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<h2 id="1">New Parts from literature:</h2> | <h2 id="1">New Parts from literature:</h2> | ||
<h3>Promoters:</h3> | <h3>Promoters:</h3> | ||
− | <p>In order to achieve robustness in the system, it is necessary to have a library of promoters with a wide range of transcription rates. One such library of synthetic promoters from Liu et al. (2018) consisted of 214 synthetic promoters with consensus sequence as shown below | + | <p>In order to achieve robustness in the system, it is necessary to have a library of promoters with a wide range of transcription rates. One such library of synthetic promoters from Liu et al. (2018) consisted of <strong>214 synthetic promoters </strong>with consensus sequence as shown below [1]:</p> |
<div class="trable-responsive py-3" style="overflow-x: scroll;"> | <div class="trable-responsive py-3" style="overflow-x: scroll;"> | ||
<img src="https://static.igem.org/mediawiki/2021/1/1a/T--IISER-Tirupati_India--SP_Backbone.jpg" alt="Letter representation of SP backbone showing different regions."> | <img src="https://static.igem.org/mediawiki/2021/1/1a/T--IISER-Tirupati_India--SP_Backbone.jpg" alt="Letter representation of SP backbone showing different regions."> | ||
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<p class="text-center">Fig. 1 SP Backbone</p> | <p class="text-center">Fig. 1 SP Backbone</p> | ||
− | <p>All these promoters are constitutive hence can be used for general protein production. From this library we used SP126, SP146 and SP200 having relative activity with respect to <a target="_blank" href="http://parts.igem.org/Part:BBa_K143013">P43</a> as follows:</p> | + | <p>All these promoters are <strong>constitutive</strong> hence can be used for general protein production. From this library we used SP126, SP146 and SP200 having relative activity with respect to <a target="_blank" href="http://parts.igem.org/Part:BBa_K143013">P43</a> as follows:</p> |
<div class="table-responsive"> | <div class="table-responsive"> | ||
<table class="table table-striped"> | <table class="table table-striped"> | ||
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<h3>P22 Operator Library:</h3> | <h3>P22 Operator Library:</h3> | ||
− | <p>P22 repressor (<a class="text-primary" target="_blank" href="http://parts.igem.org/wiki/index.php/Part:BBa_K3889020">BBa_K3889020</a>) binds to this sequence as a dimer. This inhibits the enzymes from transcribing the genes on whose promoter this operator site is fused. Hence this could be used with any promoter in order to form a repressible system. Different binding affinities of a repressor provide a variable system that can be used for different expression levels of the target thereby enabling it in a variety of systems. Optimization and tweaking of a system can be done by varying the operator sites as well.</p> | + | <p>P22 c2 repressor (<a class="text-primary" target="_blank" href="http://parts.igem.org/wiki/index.php/Part:BBa_K3889020">BBa_K3889020</a>) binds to this sequence as a dimer. This inhibits the enzymes from transcribing the genes on whose promoter this operator site is fused. Hence this could be used with any promoter in order to form a repressible system. <strong>Different binding affinities</strong> of a repressor provide a variable system that can be used for different expression levels of the target thereby enabling it in a variety of systems [2]. Optimization and tweaking of a system can be done by varying the operator sites as well.</p> |
<div class="table-responsive"> | <div class="table-responsive"> | ||
Line 410: | Line 410: | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
− | </div> | + | </div><br> |
− | + | ||
<figure class="col-12 col-sm-10 col-md-8 m-auto"> | <figure class="col-12 col-sm-10 col-md-8 m-auto"> | ||
<img class="text-primary" src="https://static.igem.org/mediawiki/2021/8/88/T--IISER-Tirupati_India--Kd_values_of_P22_binding_site.png" alt="Histogram showing the different values of K<sub>D</sub> values of different parts on the y-axis, there is rel K<sub>D</sub> and on the x-axis there is part number." style="width:100%"> | <img class="text-primary" src="https://static.igem.org/mediawiki/2021/8/88/T--IISER-Tirupati_India--Kd_values_of_P22_binding_site.png" alt="Histogram showing the different values of K<sub>D</sub> values of different parts on the y-axis, there is rel K<sub>D</sub> and on the x-axis there is part number." style="width:100%"> | ||
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<h3>Coding sequences:</h3> | <h3>Coding sequences:</h3> | ||
− | <p><a target="_blank" href="http://parts.igem.org/Part:BBa_K3889021">SRTF1</a> or steroid responsive transcription factor 1 can negatively regulate any promoter activity | + | <p><a target="_blank" href="http://parts.igem.org/Part:BBa_K3889021">SRTF1</a> or steroid responsive transcription factor 1 can negatively regulate any promoter activity if its binding site is fused with the gene's promoter. SRTF1 binds to its binding site(<a target="_blank" href="http://parts.igem.org/Part:BBa_K3889030">BBa_K3889030</a>) as done in <a target="_blank" href="http://parts.igem.org/Part:BBa_K3889150">BBa_K3889150</a>. Presence of progesterone causes unbinding of SRTF1 thereby releasing it from the DNA, inducing the target gene.Thus,progesterone acts as an inducer and can be used in a <strong>progesterone inducible system</strong> by other teams as well [3].</p> |
<p id="14"><br /><br /></p> | <p id="14"><br /><br /></p> | ||
<h3>Device:</h3> | <h3>Device:</h3> | ||
− | <p>Terminator checking device (<a target="_blank" href="http://parts.igem.org/BBa_K3889140">BBa_K3889140</a>): In order to check terminator efficiency a simple reference circuit was used similar to what used by Gale et al. (2021) | + | <p>Terminator checking device (<a target="_blank" href="http://parts.igem.org/BBa_K3889140">BBa_K3889140</a>): In order to check terminator efficiency a simple reference circuit was used similar to what used by Gale et al. (2021) [4] as shown below:</p> |
<figure class="col-12 col-sm-10 col-md-8 m-auto"> | <figure class="col-12 col-sm-10 col-md-8 m-auto"> | ||
<img src="https://static.igem.org/mediawiki/2021/6/63/T--IISER-Tirupati_India--contributiontermcheckdevice_01.jpg" alt="Genetic Circuit of terminator check device." style="width:100%"> | <img src="https://static.igem.org/mediawiki/2021/6/63/T--IISER-Tirupati_India--contributiontermcheckdevice_01.jpg" alt="Genetic Circuit of terminator check device." style="width:100%"> | ||
<figcaption class="text-center p-3"> | <figcaption class="text-center p-3"> | ||
− | Fig.3 - Terminator Check Device | + | Fig.3 - Terminator Check Device/Reference |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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</figure> | </figure> | ||
<div class="table-responsive"> | <div class="table-responsive"> | ||
− | <p>Formulae for terminator efficiency | + | <p>Formulae for terminator efficiency [4] :</p> |
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<p>\(mCherry_{0} \rightarrow\) mCherry produced by device without terminator</p><br> | <p>\(mCherry_{0} \rightarrow\) mCherry produced by device without terminator</p><br> | ||
<p>\(sfGFP_{0} \rightarrow\) sfGFP produced by device without terminator</p><br> | <p>\(sfGFP_{0} \rightarrow\) sfGFP produced by device without terminator</p><br> | ||
− | Using the device without any changes, \(TE_{Device}\) can be calculated which gives the expression of | + | Using the <strong>device/reference</strong> without any changes, \(TE_{Device}\) can be calculated which gives the expression of \(mCherry\) in absence of a terminator.<br><br> |
\(TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]\) (2)<br><br> | \(TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]\) (2)<br><br> | ||
where, <br><br> | where, <br><br> | ||
Line 601: | Line 601: | ||
</td> | </td> | ||
<td> | <td> | ||
− | <p id="16"> | + | <p id="16">Mutated Kpn1 site and stop codon</p> |
</td> | </td> | ||
</tr> | </tr> | ||
Line 610: | Line 610: | ||
<h3 class="p-3">REFERENCES</h3> | <h3 class="p-3">REFERENCES</h3> | ||
<ol> | <ol> | ||
− | <li | + | <li>Liu, D., Mao, Z., Guo, J., Wei, L., Ma, H., Tang, Y., Chen, T., Wang, Z., & Zhao, X. (2018). Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis. ACS Synthetic Biology, 7(7), 1785–1797. https://doi.org/10.1021/acssynbio.8b00115 </li> |
− | + | ||
− | + | ||
− | <li | + | <li>Watkins, D., Hsiao, C., Woods, K. K., Koudelka, G. B., & Williams, L. D. (2008). P22 c2 Repressor− Operator Complex:  Mechanisms of Direct and Indirect Readout. Biochemistry, 47(8), 2325–2338. https://doi.org/10.1021/bi701826f</li> |
− | <li | + | <li>Baer, R. Cooper (2020). Discovery, characterization, and ligand specificity engineering of a novel bacterial transcription factor inducible by progesterone Boston University School of Medicine, 801 Massachusetts Avenue Suite 400 Boston, MA 02118 Retrieved from: https://hdl.handle.net/2144/41109</li> |
− | <li | + | <li>Gale, G. A. R., Wang, B., & McCormick, A. J. (2021). Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.624011 </li> |
</ol> | </ol> | ||
Revision as of 18:05, 20 October 2021
New Parts from literature:
Promoters:
In order to achieve robustness in the system, it is necessary to have a library of promoters with a wide range of transcription rates. One such library of synthetic promoters from Liu et al. (2018) consisted of 214 synthetic promoters with consensus sequence as shown below [1]:
![Letter representation of SP backbone showing different regions.](https://static.igem.org/mediawiki/2021/1/1a/T--IISER-Tirupati_India--SP_Backbone.jpg)
Fig. 1 SP Backbone
All these promoters are constitutive hence can be used for general protein production. From this library we used SP126, SP146 and SP200 having relative activity with respect to P43 as follows:
Promotor |
Sequence 5' → 3' |
Relative activity wrt P43 - GFP (%) |
Standard deviation |
---|---|---|---|
AAAAATTATAAAAATGTGTTGACAAAGGGGGTCCTGTATGTTATAATAGCTT |
29.07 |
0.23 |
|
AAAAATAACAAAAACGTGTTGACAATAAAGATTAACCGTGATATAATTAAAT |
40.39 |
0.69 |
|
AAAAATTAGAAAAATGTGTTGACACTCGGACGAAACAATGGTATAATGGCAA |
76.82 |
0.9 |
P22 Operator Library:
P22 c2 repressor (BBa_K3889020) binds to this sequence as a dimer. This inhibits the enzymes from transcribing the genes on whose promoter this operator site is fused. Hence this could be used with any promoter in order to form a repressible system. Different binding affinities of a repressor provide a variable system that can be used for different expression levels of the target thereby enabling it in a variety of systems [2]. Optimization and tweaking of a system can be done by varying the operator sites as well.
Part Name |
Sequence |
Rel KD |
K D (in M) |
---|---|---|---|
ATTTAAGATATCTTAAAT |
1 |
1.6 × 10−8 |
|
AATTAAGATATCTTAATT |
1.8 |
2.88 × 10-8 |
|
ATTTAAGAATTCTTAAAT |
2 |
3.2 × 10−8 |
|
AGTTAAGATATCTTAACT |
2.6 |
4.16 × 10−8 |
|
ATTAAAGATATCTTTAAT |
3.8 |
6.08 × 10−8 |
|
ACTTAAGATATCTTAAGT |
4.3 |
6.88 × 10−8 |
|
ATTCAAGATATCTTGAAT |
5 |
8.0 × 10−8 |
|
ATTGAAGATATCTTCAAT |
7.6 |
1.216 × 10−7 |
|
ATTTAAGAGCTCTTAAAT |
10 |
1.6 × 10−7 |
|
ATTTAAGACGTCTTAAAT |
10 |
1.6 × 10−7 |
|
ATTTACGATATCGTAAAT |
30 |
4.8 × 10−7 |
|
ATTTAAAATATTTTAAAT |
55 |
8.8 × 10−7 |
![Histogram showing the different values of K<sub>D</sub> values of different parts on the y-axis, there is rel K<sub>D</sub> and on the x-axis there is part number.](https://static.igem.org/mediawiki/2021/8/88/T--IISER-Tirupati_India--Kd_values_of_P22_binding_site.png)
Coding sequences:
SRTF1 or steroid responsive transcription factor 1 can negatively regulate any promoter activity if its binding site is fused with the gene's promoter. SRTF1 binds to its binding site(BBa_K3889030) as done in BBa_K3889150. Presence of progesterone causes unbinding of SRTF1 thereby releasing it from the DNA, inducing the target gene.Thus,progesterone acts as an inducer and can be used in a progesterone inducible system by other teams as well [3].
Device:
Terminator checking device (BBa_K3889140): In order to check terminator efficiency a simple reference circuit was used similar to what used by Gale et al. (2021) [4] as shown below:
![Genetic Circuit of terminator check device.](https://static.igem.org/mediawiki/2021/6/63/T--IISER-Tirupati_India--contributiontermcheckdevice_01.jpg)
Now spacer can be replaced with any terminator in order to see the expression of sfGFP and mCherry.
![Genetic circuit showing the terminator to be checked.](https://static.igem.org/mediawiki/2021/c/ca/T--IISER-Tirupati_India--contributiontermtobechecked_01.jpg)
Formulae for terminator efficiency [4] :
\(TE_{Device} = \frac{mCherry_{0}}{sfGFP_{0}}\)
where,
\(mCherry_{0} \rightarrow\) mCherry produced by device without terminator
\(sfGFP_{0} \rightarrow\) sfGFP produced by device without terminator
Using the device/reference without any changes, \(TE_{Device}\) can be calculated which gives the expression of \(mCherry\) in absence of a terminator.
\(TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]\) (2)
where,
\(mCherry \rightarrow\) mCherry produced by device with the terminator that needs to checked
\(sfGFP \rightarrow\) sfGFP produced by device with the terminator that needs to checked
Modifications in the old parts:
Old part |
New part |
Name |
Modifications made |
---|---|---|---|
XylR |
Removed Dual stop codons |
||
P22 c2 repressor |
Removed Sap1 Recognition Site, LVA tag and Barcodes |
||
P2A Peptide Linker PTV |
BsmBI recognition site removed for Golden Gate compatibility |
||
YqcG toxin |
Mutated Xho1, HindIII and Bsa1 sites to make assembly compatible part |
||
YqcF antitoxin |
Mutated BgIII site |
||
YtvA |
Mutated Bsa1 and HindIII Sites |
||
bovine pancreatic DNase 1 |
Mutated HindIII Site |
||
mf-Lon protease |
Mutated multiple RE sites |
||
sfGFP |
Mutated Kpn1 site and stop codon |
REFERENCES
- Liu, D., Mao, Z., Guo, J., Wei, L., Ma, H., Tang, Y., Chen, T., Wang, Z., & Zhao, X. (2018). Construction, Model-Based Analysis, and Characterization of a Promoter Library for Fine-Tuned Gene Expression in Bacillus subtilis. ACS Synthetic Biology, 7(7), 1785–1797. https://doi.org/10.1021/acssynbio.8b00115
- Watkins, D., Hsiao, C., Woods, K. K., Koudelka, G. B., & Williams, L. D. (2008). P22 c2 Repressor− Operator Complex: Mechanisms of Direct and Indirect Readout. Biochemistry, 47(8), 2325–2338. https://doi.org/10.1021/bi701826f
- Baer, R. Cooper (2020). Discovery, characterization, and ligand specificity engineering of a novel bacterial transcription factor inducible by progesterone Boston University School of Medicine, 801 Massachusetts Avenue Suite 400 Boston, MA 02118 Retrieved from: https://hdl.handle.net/2144/41109
- Gale, G. A. R., Wang, B., & McCormick, A. J. (2021). Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species. Frontiers in Microbiology, 11. https://doi.org/10.3389/fmicb.2020.624011
Gene Gala
We held a Mini-Summer school in collaboration with the iGEM 2021 team of IISER Kolkata. It was a 5-day Mini-Summer School for Girl students studying in 12th Standards of the schools under the Directorate of Education, GNCT Delhi. As part of the summer school, the two teams together prepared a 5-days lesson plan, 2 quiz sessions, and a day-to-day handbook made for reference for the students. We would like to present these resources as a contribution to iGEM.
Future iGEM teams can use them directly for conducting similar programs in their regions/countries to the relevant audiences giving proper attributions to both the contributing teams. These resources will be extremely useful for teams who are preparing for similar education events. Conducting classes for 5 days enriched with activities and quiz sessions can be a daunting task for teams. The lesson plan provided here was able to keep the students engaged throughout the 5 days and it was easy for the team members to present as well. These content handbooks, lesson plans, and quizzes will come in handy for future iGEM teams to prepare for such an event and take their public engagement to the next level.
The content is relevant for introducing high school seniors to Synthetic Biology while giving them a holistic and application-based view of the biology courses taught at the high school level.
Downloads Mini Summer School Resources
Gene Gala-Handbook
Click here on button to download the PDF file.
-
Gene Gala Quiz 1
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-
Gene Gala Quiz 2
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-
Gene Gala Quiz Answer Key
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Lesson plan - Gene Gala
Click here on button to download the PDF file.
Class Material
Day 1
Click here on button to download the PDF file.
-
Day 2
Click here on button to download the PDF file.
Day 3
Click here on button to download the PDF file.
Day 4
Click here on button to download the PDF file.
Note : It will be helpful if 2 people present the content, which will stop the lesson from becoming monotonous and keep students engaged.
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