Part:BBa_K3889070
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Double terminator for Bacillus subtilis
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- Secondary Structure:
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].
Fig 1. Spacer Cassette for Terminator check
Fig 2. Spacer replaced by BBa_B0010
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]:
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.
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:
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:
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:
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:
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
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