Team:IISER-Tirupati India/Parts

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INDEX

• Overview
• Previous Parts
• Basic Parts
• Composite Parts
• Improvement
• References

Overview

Due to the novel approaches taken while pursuing this project, we found ourselves using new parts that had not been given in the iGEM registry. In total, we developed 37 new basic parts (either entirely new or by modifying previously existing ones). We strongly believe that these two collections can be exploited to their full potential by the subsequent iGEM generations.

Furthermore, we have designed and used multiple combinations of basic parts as given in the composite part section.These composite parts can be used in multiple settings depending on the requirements of the projects.

The parts used in our project are as follows:

Previous Parts used:

BioBrick ID	Name	Length	Description
BBa_K316001	pVeg	97 bp	Constitutive natural promoter under Sig A transcription factor
BBa_K143013	P43	56 bp	Constitutive natural promoter under Sig A and SigB transcription factor
BBa_K143014	pxylA	82 bp	Xylose dependent natural promoter (repressed by xylR)
BBa_K2333002	Protein degradation tag B	87 bp	mf-Lon specific Protein Degradation Tag B (medium-strong)
BBa_K1351028	RBS	11 bp	RBS for B. subtilis
BBa_K1351030	RBS	11 bp	RBS for B. subtilis
BBa_K1351031	RBS	11 bp	RBS for B. subtilis
BBa_K1351033	RBS	11 bp	RBS for B. subtilis
BBa_J06504	mCherry	714 bp	Red fluorescent tag
BBa_B0015	Terminator	129 bp	Double terminator consisting of BBa_B0010 and BBa_B0012
BBa_B0010	Terminator	80 bp	Relatively weaker Terminator as compared to BBa_B0015
BBa_K2314608	TMini	68 bp	Very short terminator in yeast with good performance

Basic Parts: 

Part No:	Name	Length	Description
BBa_K3889000	Azurite BFP (BFP.B3)	717 bp	Blue fluorescent tag
BBa_K3889002	modified_sfGFP	720 bp	Green fluorescent tag
BBa_K3889010	SP126	52 bp	Promoter from synthetic library of Bacillus subtilis
BBa_K3889011	SP146	52 bp	Promoter from synthetic library of Bacillus subtilis
BBa_K3889012	SP200	52 bp	Promoter from synthetic library of Bacillus subtilis
BBa_K3889013	pgsiB	300 bp	Natural promoter of Bacillus subtilis under control of general stress SigB transcription factor
BBa_K3889014	pGAL1 Yeast Promoter	531 bp	Galactose inducible yeast promoter
BBa_K3889020	P22 c2 repressor	648 bp	DNA binding that represses gene expression
BBa_K3889021	Steroid responsive Transcription Factor (SRTF1)	567 bp	Transcription factor that can bind to specific DNA sequence to repress gene expression and is inhibited by progesterone
BBa_K3889022	Human Ovastacin protease phosphomimic_A	594 bp	Epitope region of the human protease ovastacin containing active site for ZP2 protein cleavage
BBa_K3889023	Human Ovastacin protease phosphomimic_B	594 bp	Human protease ovastacin with phospho mimic tyrosine
BBa_K3889024	Human Ovastacin protease	594 bp	Human protease ovastacin with phospho mimic tyrosine and serine
BBa_K3889025	yqcG toxin	1593 bp	Toxin part of type 2 toxin-antitoxin system of Bacillus subtilis (DNase)
BBa_K3889026	ytvA light sensor	783 bp	Blue light sensor that positively regulates SigB
BBa_K3889027	bovine pancreatic DNase 1	846 bp	Highly potential and efficient endonuclease (Toxin)
BBa_K3889028	mf-Lon protease	2361 bp	Lon Protease from Mesoplasma florum bacteria
BBa_K3889029	Human Zona pellucida ZP2 protein partial	1800 bp	ZP2 protein that is cleaved by ovastacin
BBa_K3889030	SRTF 1 Binding Site	20 bp	SRTF1 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889040	Bacillus subtilis Spacer Sequence	40 bp	Spacer sequence without any promoter, RBS or terminator activity
BBa_K3889050	Bacillus subtilis TAT signal peptide PhoD	168 bp	Signal Peptide for TAT secretion system in Bacillus subtilis
BBa_K3889051	Bacillus subtilis TAT signal peptide YwbN	84 bp	Signal Peptide for TAT secretion system in Bacillus subtilis
BBa_K3889069	P2A Peptide Linker PTV	66 bp	Self cleaving peptide sequence which separates two proteins during translation in same operon
BBa_K3889070	Bacillus subtilis oriented double terminator nagP	122 bp	Fused product of nagP and BBa_B0010 (Improvement in BBa_B0010)
BBa_K3889080	P22 binding site A	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889081	P22 binding site B	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889082	P22 binding site C	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889083	P22 binding site D	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889084	P22 binding site E	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889085	P22 binding site F	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889086	P22 binding site G	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889087	P22 binding site H	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889088	P22 binding site I	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889089	P22 binding site J	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889090	P22 binding site K	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889091	P22 binding site L	18 bp	P22 binds to this DNA sequence to negatively regulate gene expression
BBa_K3889092	XylR	1050 bp	pXylA repressor which provides xylose inducible gene expression
BBa_K3889093	yqcF	576 bp	Anti-toxin of yqcG (a kind of DNase)

Composite Parts: 

Part No:	Name	Length	Description
BBa_K3889100	SRTF1 expression cassette	1539 bp	Constitutive production of SRTF1 being reported by mCherry
BBa_K3889101	P22 expression cassette	1643 bp	Progesterone inducible production due to fusion of SRTF1 binding site to the promoter reported by Azurite
BBa_K3889102	Ovastacin expression cassette	1042 bp	P22 controlled (indirect progesterone control) ovastacin production reported by sfGFP
BBa_K3889110	XylR Repressor Cassette	2032 bp	Constitutive production of XylR being reported by sfGFP
BBa_K3889111	yqcG toxin casette	1815 bp	Xylose inducible production of YqcG
BBa_K3889112	yqcF antitoxin cassette	1548 bp	Constitutive production of YqcF being reported by mCherry
BBa_K3889120	YtvA expression cassette	1759 bp	Constitutive production of YtvA being reported by mCherry
BBa_K3889121	bovine pancreatic DNase 1 toxin cassette	1275 bp	Light inducible production of bpDNase 1
BBa_K3889122	mf-Lon cassette	3339 bp	Constitutive production of mf-Lon being reported by sfGFP
BBa_K3889127	bpDNase1+mfLon_pdtB	933 bp	bpDnase 1 fused with protein degradation tag of mf-Lon
BBa_K3889130	Spacer Cassette for Terminator check in Bacillus subtilis	1722 bp	Spacer in between of two reporters mCherry and sfGFP which provides with basal level expression of downstream genes in absence of a terminator
BBa_K3889131	BBa_B0010 terminator check cassette	1762 bp	Replacing spacer with terminator in BBa_K3889130 for checking terminator efficiency of BBa_B0010
BBa_K3889132	BBa_B0010-nagP fused terminator check cassette	1804 bp	Replacing spacer with terminator in BBa_K3889130 for checking terminator efficiency of BBa_K3889070
BBa_K3889150	SP200+SRTF1 Binding Site+ RBS	83 bp	SRTF1 binding site fused with SP200 promoter for progesterone inducible gene expression
BBa_K3889151	P43+P22 Binding Site L +RBS	85 bp	P22 binding site fused with P43 for P22 controlled gene expression

Improvement:

Introduction:

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 Reference

Fig 2. Spacer replaced by BBa_B0010

Fig 3. Spacer replaced by BBa_K3889070

The device/reference (Fig 1.) and the test cassette (Fig 2 and 3.) provide us 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]

\begin{equation}\tag{1}TE_{Device}=\frac{mCherry_{0}}{sfGFP_{0}}\end{equation}
where,

$mCherry_{0}\rightarrow$ mCherry produced by device without terminator

$sfGFP_{0}\rightarrow$ sfGFP produced by device without terminator

Using the device without any changes, $TE_{Device}$ can be calculated which gives the expression of
$mCherry$ in absence of a terminator.

\begin{equation}\tag{2} TE=100-\left[\left(\frac{mCherry}{sfGPF}\right)\times\left(\frac{1}{TE_{Device}}\right)\times100\right]\end{equation}

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

d-score:

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

$d=96.59 \times \frac{-\Delta G/(kcal/mol)}{n_{SL}} + 18.16 \times T_{score} -116.87$

Where 

d is the d-score

$-\Delta G$ is the Gibbs free energy of stem-loop formation in kcal/mole

n_SL is the length of the stem loop

T_Score 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:

$T_{score}= \sum\limits_{i=0}^{\ 14} x _i$

Where

$x_0 = 0.9$

$x_i = 0.9$ if $i^{th}$ nucleotide is thymine

$x_i = 0.6 \times x_{i-1}$ if $i^{th}$ nucleotide is not thymine

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

$d=7.90 \times \frac{-\Delta G/(kcal/mol)}{n_{SL}} + 2.67 \times T_{score} -14.91$

Where 

d is the d-score

$-\Delta$ G is the Gibbs free energy of stem-loop formation in kcal/mole

n _SL is the length of the stem loop

T_Score is the score for T-stretch of the terminators

Coefficients are according to fitting the model 

Here the T_Score is calculated as follows:

$T= \sum\limits_{i=0}^{\ 14} e^{- \lambda _i} \delta_i$

Where

$\lambda _i = 0.144$ as per the fitting of the model

$\delta_i = 0$ if $i^{th}$ nucleotide is not thymine 

$\delta_i = 1$ if $i^{th}$ 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 B. 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. 

	BBa_B0010	BBa_B0010+nagP
Minimum Free Energy (kcal/mol)	-40	-64.6

The predicted structure for these two terminators as given by RnaFold server is:

1. BBa_B0010:

   

   Fig 4. BBa_B0010 from RnaFold

2. BBa_B0010+nagP:

   

   Fig 5. Fused BBa_B0010+nagP from RnaFold

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 

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