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| | Premature termination was avoided and theoretical efficiency was taken into consideration while designing. | | Premature termination was avoided and theoretical efficiency was taken into consideration while designing. |
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| − | <h3>Overview </h3>
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| − | 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.
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| − | <h3>Measuring efficiency:</h3>
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| − | 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].
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| − | [[File:T--IISER-Tirupati India--Improvement1.jpeg|690px]]<br>
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| − | Fig 1. Spacer Cassette for Terminator check
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| − | [[File:T--IISER-Tirupati India--Improvement2.jpeg|690px]]<br>
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| − | Fig 2. Spacer replaced by BBa_B0010
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| − | [[File:T--IISER-Tirupati India--Improvement3.jpeg|690px]]<br>
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| − | Fig 3. Spacer replaced by BBa_K3889070
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| − | 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.
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| − | Formulae for terminator efficiency [1]:
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| − | [[File:T--IISER-Tirupati_India--Eq1.png]]
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| − |
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| − | where
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| − | mCherry<sub>0</sub> → mCherry produced by device without terminator
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| − | sfGFP<sub>0</sub> → sfGFP produced by device without terminator
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| − |
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| − | Using the device without any changes, TE<sub>Device</sub> can be calculated which gives the expression of mCherry in absence of a terminator.
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| − |
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| − | [[File:T--IISER-Tirupati_India--Eq2.png]]
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| − |
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| − | where
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| − | mCherry → mCherry produced by the device with terminator
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| − | sfGFP → sfGFP produced by the device with terminator
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| − | <h3>d-score</h3>
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| − |
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| − | For <i>E. coli</i> terminators d'Aubenton Carafa [3] gave a scoring system as shown below:
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| − |
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| − | [[File:T--IISER-Tirupati_India--Eq3.png]]
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| − |
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| − | where
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| − | d is the d-score
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| − | −ΔG is the Gibbs free energy of stem-loop formation in kcal/mole
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| − | n<sub>SL</sub> is the length of the stem loop
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| − | T<sub>Score</sub> is the score for T-stretch of the terminators
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| − |
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| − | Coefficients are according to fitting the d'Aubenton Carafa’s model
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| − |
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| − | The T-Score is calculated as follows:
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| − |
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| − | [[File:T--IISER-Tirupati_India--Eq5.png]]
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| − |
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| − | where
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| − | x<sub>0</sub> = 0.9
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| − | x<sub>i</sub> = 0.9 if i<sup>th</sup> nucleotide is thymine
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| − |
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| − | x<sub>i</sub> = 0.6*x<sub>i-1</sub> if i<sup>th</sup> nucleotide is not thymine
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| − |
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| − | 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:
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| − |
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| − | [[File:T--IISER-Tirupati_India--Eq6.png]]
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| − |
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| − | where
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| − |
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| − | d is the d-score
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| − |
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| − | −ΔG is the Gibbs free energy of stem-loop formation in kcal/mole
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| − |
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| − | n<sub>SL</sub> is the length of the stem loop
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| − |
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| − | T<sub>Score</sub> is the score for T-stretch of the terminators
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| − |
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| − | Coefficients are according to fitting the de Hoon et al. model
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| − |
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| − | Here, the T-Score can be calculated as follows:
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| − |
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| − | [[File:T--IISER-Tirupati_India--Eq4.png]]
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| − |
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| − | where
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| − | λ<sub>i</sub> = 0.144 as per de Hoon et al. model
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| − |
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| − | δ<sub>i</sub> = 0 if i<sup>th</sup> nucleotide is not thymine
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| − |
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| − | δ<sub>i</sub> = 1 if i<sup>th</sup> nucleotide is thymine
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| − |
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| − | 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]
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| − |
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| − | <h3>Improvement</h3>
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| − | 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.
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| − | 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.
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| − |
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| − | 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]
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| − | {| class="wikitable"
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| − | !<partinfo>BBa_B0010</partinfo>
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| − | !<partinfo>BBa_K3889070</partinfo>
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| − | |-
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| − | !Minimum Free Energy (kcal/mol)
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| − | -40.0
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| − | -64.6
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| − | |}
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| | Bacillus subtilis genome and iGEM registry | | Bacillus subtilis genome and iGEM registry |
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| − | ===References===
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| − | 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 :
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| − | 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
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| − | 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
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| − | 4. http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi
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Premature termination was avoided and theoretical efficiency was taken into consideration while designing.
