Difference between revisions of "Part:BBa K3889070"
 
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'''Secondary Structure'''
+
*Secondary Structure:
  
−
[[Image:Mfold-K3889070-1.png]]
+
[[File:Mfold-K3889070-1.png]]
  
−
<hr>
+
<h3>Overview </h3>
−
'''Measurement'''
+
While engineering any new circuit, there is always a need for well-characterized and predictable parts. Not only should the circuit function as expected, but it should also be orthogonal to irrelevant cell processes, thereby increasing the need to have efficient production and, in some cases, more importantly, efficient termination.  While there are several well-studied and efficient terminators for <i>E. coli</i>, we found no specific efficient single terminator on the iGEM registry that could stand out for <i>B.subtilis</i> chassis. Hence, we decided to improvise a terminator which might fulfil this gap.
−
* [http://openwetware.org/wiki/Cconboy:Terminator_Characterization/Results How these parts were measured]
+
  
−
<html>
+
<h3>Measuring efficiency:</h3>
−
<body>
+
The experiment is divided into two cassettes: one reference and the other is a test cassette containing a terminator whose efficiency needs to be determined as shown by Gale et al.[1].
−
<h1>Improvement:</h1>
+
  
−
                            <p><strong>Introduction:</strong></p>
+
[[File:T--IISER-Tirupati India--Improvement1.jpeg|690px]]<br>
−
                           
+
Fig 1. Spacer Cassette for Terminator check
−
                            <p>While engineering any new circuit, there is always a need for well-characterized and predictable parts. Not only should the circuit function as expected, but it should also be orthogonal to irrelevant cell processes, thereby increasing the need to have efficient production and, in some cases, more importantly, efficient termination. While there are several well-studied and efficient terminators for <em>E.coli</em>, we found no specific efficient single terminator on the iGEM registry that could stand out for <em>B.subtilis</em> chassis. Hence, we decided to improvise a terminator which might fulfil this gap.</p>
+
  
−
                            <p><strong>Measuring efficiency:</strong></p>
+
[[File:T--IISER-Tirupati India--Improvement2.jpeg|690px]]<br>
−
                            <p>The experiment is divided into two cassettes: one reference and the other is a test cassette containing a terminator whose efficiency needs to be determined as shown by Gale et al.[1].</p>
+
Fig 2. Spacer replaced by BBa_B0010
  
−
                            <figure class="col-12 col-md-8 p-5 m-auto">
+
[[File:T--IISER-Tirupati India--Improvement3.jpeg|690px]]<br>
−
                                <img src="https://2021.igem.org/wiki/images/b/b7/T--IISER-Tirupati_India--Improvement1.jpeg" alt="Spacer Cassette for Terminator check" style="width:100%">
+
Fig 3. Spacer replaced by BBa_K3889070
−
                                <figcaption>Fig 1. Spacer Cassette for Terminator check</figcaption>
+
−
                            </figure>
+
  
−
                            <figure class="col-12 col-md-8 p-5 m-auto">
+
The reference(Fig 1) and the test cassette (Fig 2 and 3) provide us with the expression levels of both the fluorescent proteins which could be compared to tell us how efficiently the terminator is working.
−
                                <img src="https://2021.igem.org/wiki/images/a/a7/T--IISER-Tirupati_India--Improvement2.jpeg" alt="Spacer replaced by BBa_B0010  " style="width:100%">
+
−
                                <figcaption>Fig 2. Spacer replaced by BBa_B0010</figcaption>
+
−
                            </figure>
+
−
                            <figure class="col-12 col-md-8 p-5 m-auto">
+
−
                                <img src="https://2021.igem.org/wiki/images/e/e7/T--IISER-Tirupati_India--Improvement3.jpeg" alt="Spacer replaced by BBa_K3889070" style="width:100%">
+
−
                                <figcaption>Fig 3. Spacer replaced by BBa_K3889070</figcaption>
+
−
                            </figure>                                 
+
−
                            <p>The reference and the test cassette provide us the expression levels of both the fluorescent proteins which could be compared to tell us how efficiently the terminator is working.</p>
+
−
                            <p><br /><br /></p>
+
−
                            <p>Formulae for terminator efficiency [1] </p>
+
  
−
                            \begin{equation}\tag{1}TE_{Device}=\frac{mCherry_{0}}{sfGFP_{0}}\end{equation}   
+
Formulae for terminator efficiency [1]:
−
                            <br>
+
−
                            where,
+
−
                            <br>
+
−
                            <br>
+
−
                            $mCherry_{0}\rightarrow$  mCherry produced by device without terminator<br><br>
+
−
                            $sfGFP_{0}\rightarrow$ sfGFP produced by device without terminator<br><br>
+
−
                            Using the device without any changes, $TE_{Device}$  can be calculated which gives the expression of <br>$mCherry$ in absence of a terminator.<br><br>
+
−
                            \begin{equation}\tag{2} TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]\end{equation}        <br><br>
+
−
                            where, <br><br>
+
−
                            $mCherry$ $\rightarrow$ mCherry produced by device with the terminator that needs to checked<br><br>
+
−
                            $sfGFP$ $\rightarrow$ sfGFP produced by device with the terminator that needs to checked<br><br>
+
−
                            <p><br /><br /></p>
+
−
                            <p><strong>d-score:</strong></p>
+
−
                            <p>For <em>E. coli </em>terminators d'Aubenton Carafa [3] gave a scoring system as shown below:</p>
+
  
−
                            <p>$d=96.59 \times \frac{-\Delta G/(kcal/mol)}{n_{SL}} + 18.16 \times T_{score} -116.87$</p>
+
[[File:T--IISER-Tirupati_India--Eq1.png]]
  
−
                            <p>Where&nbsp;</p>
+
where
−
                            <p>d is the d-score</p>
+
−
                            <p>$-\Delta G$ is the Gibbs free energy of stem-loop formation in kcal/mole</p>
+
−
                            <p>n<sub>SL</sub> is the length of the stem loop</p>
+
−
                            <p>TScore is the score for T-stretch of the terminators</p>
+
−
                            <p>Coefficients are according to fitting the d'Aubenton Carafa&rsquo;s model&nbsp;</p>
+
  
−
                            <p>The T<sub>Score</sub> is calculated as follows:</p>
+
mCherry<sub>0</sub> &#8594; mCherry produced by device without terminator
−
                            <p>$T_{score}= \sum\limits_{i=0}^{\ 14} x _i$</p>
+
−
                            <p>Where</p>
+
−
                            <p>$x_0 = 0.9$</p>
+
−
                            <p>$x_i = 0.9$ if $i^{th}$ nucleotide is thymine</p>
+
−
                            <p>$x_i = 0.6 \times x_{i-1}$ if $i^{th}$ nucleotide is not thymine</p>
+
  
−
                            <p>These scoring system was modified by de Hoon et al. [2] for <em>Bacillus subtilis</em> as per their model which is as follows:</p>
+
sfGFP<sub>0</sub> &#8594; sfGFP produced by device without terminator
  
−
                            <p>$d=7.90 \times \frac{-\Delta G/(kcal/mol)}{n_{SL}} + 2.67 \times T{score} -14.91$</p>
+
Using the device without any changes, TE<sub>Device</sub> can be calculated which gives the expression of mCherry in absence of a terminator.
  
−
                            <p>Where&nbsp;</p>
+
[[File:T--IISER-Tirupati_India--Eq2.png]]
−
                            <p>d is the d-score</p>
+
−
                            <p>$-\Delta$ G is the Gibbs free energy of stem-loop formation in kcal/mole</p>
+
−
                            <p>n <sub>SL</sub> is the length of the stem loop</p>
+
−
                            <p>T<sub>Score</sub> is the score for T-stretch of the terminators</p>
+
−
                            <p>Coefficients are according to fitting the model&nbsp;</p>
+
−
                            <p>Here the TScore is calculated as follows:</p>
+
  
−
                            <p>$T= \sum\limits_{i=0}^{\ 14} e^{- \lambda _i} \delta_i$</p>
+
where
−
                            <p>Where</p>
+
−
                            <p>$\lambda _i = 0.144$ as per the fitting of the model</p>
+
−
                            <p>$\delta_i = 0$ if $i^{th}$ nucleotide is not thymine&nbsp;</p>
+
−
                            <p>$\delta_i = 1$ if $i^{th}$ nucleotide is thymine</p>
+
  
−
                            <p>.As the d-score takes into account the Gibbs free energy, length of the stem-loop and the richness of thymine in the T-stretch which are essential for a rho independent terminator. Hence, the d-score can provide a rough idea about how good a terminator is. In other words, the higher the d-score higher will be the terminator efficiency.[3]</p>
+
mCherry &#8594; mCherry produced by the device with terminator
−
                            <p><br /><br /></p>
+
−
                            <p><strong>Improvement:</strong></p>
+
−
                            <p>We decided to improve BBa_B0010 in order to make a strong terminator which can be used for primarily the B.subtilis chassis while still retaining its efficiency in E.coli. For doing this we modified the tail of Bba_B0010 and fused another rho-independent terminator from the Bacillus subtilis genome on the basis of its d-Score.&nbsp;</p>
+
  
−
                            <p>From a list of 425 native B.subtilis terminators taken from the study conducted by Michiel et al [2], we calculated the d-score of each terminator to get a rough idea of their efficiency which is in the <a href="https://2021.igem.org/wiki/images/d/d8/T--IISER-Tirupati_India--improvement.zip" style="color:#0645AD;"> Data file </a> containing both data as well as T-stretch calculator python file. Based on the results the highest d-score= 5.666126119 was of the terminator belonging to the gene nagP. Both BBa_B0010 and nagP terminators were ligated to form a double terminator.</p>
+
sfGFP &#8594; sfGFP produced by the device with terminator
  
−
                            <p>Based on our calculations, we decided to go with nagP terminator. We modified the end regions of BBa_B0010 and ligated to it the nagP terminator to create an improved version(<a href="https://parts.igem.org/Part:BBa_K3889070" style="color:#0645AD;">BBa_K3889070</a>). Using the server <a href="http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi" style="color:#0645AD;">RNAFold</a> we calculated the minimum energy to show in silico that the improved terminator will have more negative Minimum Free energy as shown.&nbsp;</p>
+
and TE &#8594; is the terminator efficiency of the terminator
 +
<h3>d-score</h3>
  
−
                            <table class="table table-bordered table-striped">
+
For <i>E. coli</i> terminators d'Aubenton Carafa [3] gave a scoring system as shown below:
−
                            <thead>
+
 
−
                            <tr style="background-color: #8D1063; color: #FDF8D7;">
+
[[File:T--IISER-Tirupati_India--Eq3.png]]
−
                            <td></td>
+
 
−
                            <td>
+
where
−
                            <p>BBa_B0010</p>
+
 
−
                            </td>
+
d is the d-score
−
                            <td>
+
 
−
                            <p>BBa_B0010+nagP</p>
+
−ΔG is the Gibbs free energy of stem-loop formation in kcal/mole
−
                            </td>
+
 
−
                            </tr>
+
n<sub>SL</sub> is the length of the stem loop
−
                            </thead>
+
 
−
                            <tbody>
+
T<sub>Score</sub> is the score for T-stretch of the terminators
−
                            <tr>
+
 
−
                            <td>
+
Coefficients are according to fitting the d'Aubenton Carafa’s model
−
                            <p>Minimum Free Energy (kcal/mol)</p>
+
 
−
                            </td>
+
The T-Score is calculated as follows:
−
                            <td>
+
 
−
                            <p>-40</p>
+
[[File:T--IISER-Tirupati_India--Eq5.png]]
−
                            </td>
+
 
−
                            <td>
+
where
−
                            <p>-64.6</p>
+
 
−
                            </td>
+
x<sub>0</sub> = 0.9
−
                            </tr>
+
 
−
                            </tbody>
+
x<sub>i</sub> = 0.9 if i<sup>th</sup> nucleotide is thymine
−
                            </table>
+
 
−
</body>
+
x<sub>i</sub> = 0.6*x<sub>i-1</sub> if i<sup>th</sup> nucleotide is not thymine
−
</html>
+
 
 +
This scoring system was modified by de Hoon et al. (2005) [2] for <i>Bacillus subtilis</i> as per their model which is as follows:
 +
 
 +
[[File:T--IISER-Tirupati_India--Eq6.png]]
 +
 
 +
where
 +
 
 +
d is the d-score
 +
 
 +
−ΔG is the Gibbs free energy of stem-loop formation in kcal/mole
 +
 
 +
n<sub>SL</sub> is the length of the stem loop
 +
 
 +
T<sub>Score</sub> is the score for T-stretch of the terminators
 +
 
 +
Coefficients are according to fitting the de Hoon et al. model
 +
 
 +
Here, the T-Score can be calculated as follows:
 +
 
 +
[[File:T--IISER-Tirupati_India--Eq4.png]]
 +
 
 +
where
 +
 
 +
&lambda;<sub>i</sub> = 0.144 as per de Hoon et al. model
 +
 
 +
&delta;<sub>i</sub> = 0 if i<sup>th</sup> nucleotide is not thymine
 +
 
 +
&delta;<sub>i</sub> = 1 if i<sup>th</sup> nucleotide is thymine
 +
 
 +
As the d-score takes into account the Gibbs free energy, length of the stem-loop and the richness of thymine in the T-stretch which are essential for a rho independent terminator. Hence, the d-score can provide a rough idea about how good a terminator is. In other words, the higher the d-score higher will be the terminator efficiency.[3]
 +
 
 +
<h3>Improvement</h3>
 +
 
 +
We decided to improve BBa_B0010 in order to make a strong terminator which can be used for primarily the <i>B.subtilis</i> chassis while still retaining its efficiency in <i>E.coli</i>. For doing this we modified the tail of Bba_B0010 and fused another Rho-independent terminator from the <i>Bacillus subtilis</i> genome on the basis of its d-Score.
 +
 
 +
From a list of 425 native <i>B. subtilis</i> terminators taken from the study conducted by de Hoon et al [2], we calculated the d-score of each terminator to get a rough idea of their efficiency which is in the [[Media:Data file.zip|data file]] containing both data as well as T-stretch calculator python file. Based on the results the highest d-score= 5.666126119 was of the terminator belonging to the gene nagP. Both <partinfo>BBa_B0010</partinfo> and nagP terminators were ligated to form a double terminator.
 +
 
 +
Based on our calculations, we decided to go with nagP terminator. We modified the end regions of <partinfo>BBa_B0010</partinfo> and ligated to it the nagP terminator to create an improved version(<partinfo>BBa_K3889070</partinfo>). Using the server RNAFold we calculated the minimum energy to show in silico that the improved terminator will have more negative Minimum Free energy as shown.[4]
 +
{| class="wikitable"
 +
|
 +
!<partinfo>BBa_B0010</partinfo>
 +
!<partinfo>BBa_K3889070</partinfo>
 +
|-
 +
!Minimum Free Energy (kcal/mol)
 +
|
 +
-40.0
 +
|
 +
-64.6
 +
|}
 +
 
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K3889070 SequenceAndFeatures</partinfo>
 +
 
 +
 
 +
===References===
 +
1. 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 
 +
 
 +
2. de Hoon, M. J. L., Makita, Y., Nakai, K., & Miyano, S. (2005). Prediction of Transcriptional Terminators in Bacillus subtilis and Related Species. PLoS Computational Biology, 1(3), e25. https://doi.org/10.1371/journal.pcbi.0010025
 +
 
 +
3. Carafa, Y. d’Aubenton, Brody, E., & Thermes, C. (1990). Prediction of rho-independent Escherichia coli transcription terminators. Journal of Molecular Biology, 216(4), 835–858. https://doi.org/10.1016/s0022-2836(99)80005-9
 +
 
 +
4. http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi

Latest revision as of 19:50, 18 October 2021


Terminator.png

Double terminator for Bacillus subtilis

  • Double terminator fusing B0010 aand nagP native terminator for use in Bacillus subtilis.
  • Secondary Structure:

Mfold-K3889070-1.png

Overview

While engineering any new circuit, there is always a need for well-characterized and predictable parts. Not only should the circuit function as expected, but it should also be orthogonal to irrelevant cell processes, thereby increasing the need to have efficient production and, in some cases, more importantly, efficient termination. While there are several well-studied and efficient terminators for E. coli, we found no specific efficient single terminator on the iGEM registry that could stand out for B.subtilis chassis. Hence, we decided to improvise a terminator which might fulfil this gap.

Measuring efficiency:

The experiment is divided into two cassettes: one reference and the other is a test cassette containing a terminator whose efficiency needs to be determined as shown by Gale et al.[1].

T--IISER-Tirupati India--Improvement1.jpeg
Fig 1. Spacer Cassette for Terminator check

T--IISER-Tirupati India--Improvement2.jpeg
Fig 2. Spacer replaced by BBa_B0010

T--IISER-Tirupati India--Improvement3.jpeg
Fig 3. Spacer replaced by BBa_K3889070

The reference(Fig 1) and the test cassette (Fig 2 and 3) provide us with the expression levels of both the fluorescent proteins which could be compared to tell us how efficiently the terminator is working.

Formulae for terminator efficiency [1]:

T--IISER-Tirupati India--Eq1.png

where

mCherry0 → mCherry produced by device without terminator

sfGFP0 → sfGFP produced by device without terminator

Using the device without any changes, TEDevice can be calculated which gives the expression of mCherry in absence of a terminator.

T--IISER-Tirupati India--Eq2.png

where

mCherry → mCherry produced by the device with terminator

sfGFP → sfGFP produced by the device with terminator

and TE → is the terminator efficiency of the terminator

d-score

For E. coli terminators d'Aubenton Carafa [3] gave a scoring system as shown below:

T--IISER-Tirupati India--Eq3.png

where

d is the d-score

−ΔG is the Gibbs free energy of stem-loop formation in kcal/mole

nSL is the length of the stem loop

TScore is the score for T-stretch of the terminators

Coefficients are according to fitting the d'Aubenton Carafa’s model

The T-Score is calculated as follows:

T--IISER-Tirupati India--Eq5.png

where

x0 = 0.9

xi = 0.9 if ith nucleotide is thymine

xi = 0.6*xi-1 if ith nucleotide is not thymine

This scoring system was modified by de Hoon et al. (2005) [2] for Bacillus subtilis as per their model which is as follows:

T--IISER-Tirupati India--Eq6.png

where

d is the d-score

−ΔG is the Gibbs free energy of stem-loop formation in kcal/mole

nSL is the length of the stem loop

TScore is the score for T-stretch of the terminators

Coefficients are according to fitting the de Hoon et al. model

Here, the T-Score can be calculated as follows:

T--IISER-Tirupati India--Eq4.png

where

λi = 0.144 as per de Hoon et al. model

δi = 0 if ith nucleotide is not thymine

δi = 1 if ith nucleotide is thymine

As the d-score takes into account the Gibbs free energy, length of the stem-loop and the richness of thymine in the T-stretch which are essential for a rho independent terminator. Hence, the d-score can provide a rough idea about how good a terminator is. In other words, the higher the d-score higher will be the terminator efficiency.[3]

Improvement

We decided to improve BBa_B0010 in order to make a strong terminator which can be used for primarily the B.subtilis chassis while still retaining its efficiency in E.coli. For doing this we modified the tail of Bba_B0010 and fused another Rho-independent terminator from the Bacillus subtilis genome on the basis of its d-Score.

From a list of 425 native B. subtilis terminators taken from the study conducted by de Hoon et al [2], we calculated the d-score of each terminator to get a rough idea of their efficiency which is in the data file containing both data as well as T-stretch calculator python file. Based on the results the highest d-score= 5.666126119 was of the terminator belonging to the gene nagP. Both BBa_B0010 and nagP terminators were ligated to form a double terminator.

Based on our calculations, we decided to go with nagP terminator. We modified the end regions of BBa_B0010 and ligated to it the nagP terminator to create an improved version(BBa_K3889070). Using the server RNAFold we calculated the minimum energy to show in silico that the improved terminator will have more negative Minimum Free energy as shown.[4]

BBa_B0010 BBa_K3889070
Minimum Free Energy (kcal/mol)

-40.0

-64.6

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1. 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

2. de Hoon, M. J. L., Makita, Y., Nakai, K., & Miyano, S. (2005). Prediction of Transcriptional Terminators in Bacillus subtilis and Related Species. PLoS Computational Biology, 1(3), e25. https://doi.org/10.1371/journal.pcbi.0010025

3. Carafa, Y. d’Aubenton, Brody, E., & Thermes, C. (1990). Prediction of rho-independent Escherichia coli transcription terminators. Journal of Molecular Biology, 216(4), 835–858. https://doi.org/10.1016/s0022-2836(99)80005-9

4. http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi