Team:IISER-Tirupati India/Parts


Ovi-Cloak

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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].

Spacer Cassette for Terminator check
Fig 1. Spacer Cassette for Reference
Spacer replaced by BBa_B0010
Fig 2. Spacer replaced by BBa_B0010
Spacer replaced by BBa_K3889070
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

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 TScore 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

TScore is the score for T-stretch of the terminators

Coefficients are according to fitting the model 

Here the TScore 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:
    BBa_B0010 from RnaFold
    Fig 4. BBa_B0010 from RnaFold
  2. BBa_B0010+nagP:
    Fused BBa_B0010+nagP from RnaFold
    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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