Team:IISc-Bangalore/Design

Design

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CcaSR System
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Introduction

CcaSR is a much worked upon and researched optogenetic system. The CcaS/CcaR system utilizes a green-light responsive photo-sensing histidine kinase CcaS from cyanobacteria Synechocystis sp. PCC 6803 and its cognate downstream regulator CcaR. The CcaS/CcaR is used to induce the expression of a phycobilisome linker gene (cpcG2) in cyanobacteria.

Hence, if we place a gene of interest downstream of Pcpcg2 (the promoter for the optogenetic control of CpcG2 protein in Synechocystis), we can control the transcription of the gene optically. In the wild type protein expression system, the transcription levels increase 6-fold upon shining green light (as compared to the state where the red light is being used).

Molecular mechanism of control

It has been shown that there exist two different conformations of the CcaS protein, red-absorbing form (Pr, λmax = 672 nm) and green-absorbing form (Pg, λmax = 535 nm). Mass Spectrometry along with denaturation has shown that there is a double bond configurational change in the C-15 carbon (proline) from Z to E when frequency corresponding to green light is shone upon the cell. This change happens in the GAF domain of CcaS, which is the primary photo-sensing domain of the protein.

Cartoon working of CcaSR

Fig.1 Molecular Mechanism of CcaSR
Courtesy: Schmidl et al (2014)

This change in the protein structure results in the autophosphorylation of the histidine kinase domain of CcaS cyanobacteriochrome. This helps in the phosphotransfer to the cognate response regulator, CcaR protein. The phosphorylated CcaR protein can now dynamically increase the transcription by the PcpcpG2 promoter, causing a 6-fold increase (in the wild type protein).
Further, Heme Oxygenase (coded by ho1 gene) catalyzes the conversion of the Heme into Biliverdin IX Alpha with the use of a ferredoxin cofactor. This represents the first two steps involved in the biosynthesis of Phycocyanobilins (PCB). Also, enzyme phycocyanobilin ferredoxin oxidoreductase (coded by pcyA gene) catalyzes the four-electron reduction of biliverdin IX alpha to phycocyanobilin, the immediate precursor of cyanobacterial phytochromes.

CcaS v2.0 and v3.0 (mini CcaS)

Ong. et al (2018) have done research in increasing the dynamic range of the natural system and reducing the leaky character of the promoter. They were successful in creating CcaSR v2.0, in which they removed a putative unregulated transcriptional region in the PcpcG2 promoter, which resulted in 17 times less leakiness and 110 folds dynamic range.

mini-CcaS

Fig.2: Engineered mini-CcaS system.
A) Wild type CcaS with regions deleted for each version of mini-CcaS. B) Gene construct of plasmids used for experiments by Ong et al.
C) Respective transcription activity of each system wrt green and red frequencies. D) Dynamic range offered by each system.
Courtesy Ong et al (2018)

Along these lines, Sode and co-workers created a version of CcaS called mini-CcaS, in which they deleted two Per/Arnt/Sim (PAS) domains of unknown function. This when measured using Fluorimetry with sfgfp as a marker, revealed that the leakiness decreased 4-fold, and the dynamic range increased 593-fold.

Our Constructs

The following constructs were designed by us for modified CcaSR system. All of them were codon optimized for E. coli.



Fig.3 - mini-CcaS with Gp2 under modified promoter. (BBa_K3165039)



Fig.4 - mini-CcaS with ECFP under modified promoter. (BBa_K3165041)
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Fig.5 - CcaR along with Ho1 gene and PcyA gene. (BBa_K3165036)

It is noteworthy that we used mini-CcaS and PcpcG2-172, which is the modified promoter lacking the G-box created by Sodo et al. This was to ensure maximal dynamic range and minimal leaky transcription.

References

  1. Yuu Hirose, Takashi Shimada, Rei Narikawa, Mitsunori Katayama and Masahiko Ikeuchi "Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein"
  2. Keiko Mukougawa, Hirosuke Kanamoto, Toshikazu Kobayashi, Akiho Yokota, Takayuki Kohchi "Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli"
  3. Nicholas T. Ong and Jeffrey J. Tabor "A miniaturized Escherichia coli Green light sensor with high dynamic range"
  4. Zedao Liu, Jizhong Zhang, Jiao Jin, Quanfeng Liang, Qingsheng Qi "Programming Bacteria with light - Sensors and applications in synthetic biology"
  5. Oksana Polesskaya, Ancha Baranova, Sarah Bui, Nikolai Kondratev, Evgeniya Kananykhina, Olga Nazarenko, Tatyana Shapiro, Frances Barg Nardia, Vladmir Kornienko, Vikas Chandoke, Istvan Stadler, Raymond Lanzafame and Maz Myakishev-Rempel "Optogenetic regulation of transcription"
  6. Sebastian R. Schmidl, Ravi U. Sheth, Andrew Wu and Jeffrey J. Tabor "Refractoring and optimization of light-switchable Escherichia coli Two-component Systems

Opto-T7 System
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Opto-T7 RNA Polymerase

Overview

Light can be used as a good control input for biological systems due to its precise spatiotemporal resolution. Here, we present the engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-folds upon activation.

Native T7 RNA Polymerase

T7 DNA-dependent RNA Polymerase (T7RNAP), originating from the T7 bacteriophage is commonly used for protein overexpression. This Polymerase shows high processivity and high selectivity for the T7 promoter, and it does not transcribe sequences from endogenous Escherichia coli DNA. As T7RNAP-driven transcription is independent of the native E. coli RNAP, it allows inhibition of the native transcription machinery (e.g., with rifampicin), without affecting the orthogonal T7 transcription system, resulting in exclusive expression of T7RNAP expressed genes.

Making OPTO-T7 RNA Polymerase

Studies have shown that T7RNAP can be split and reconstituted through dimerization to enhance and control its function. The T7RNAP is made light-inducible by splitting the Polymerase into two fragments and fusing them to photoactivable dimerization domains. For our project, we used the heterodimeric “Magnet” domains to implement light control. This is analogous to the well-established BiFC (Bimolecular Fluorescence Complementation) technique. Magnets are derived from the small homo-dimerizing photoreceptor Vivid (VVD) from the filamentous fungus Neurospora crassa and consist of the nMag and pMag heterodimerizing protein domains, which specifically bind to each other. Magnets use flavin as a chromophore for blue light- induced binding of the two domains, which is abundant in bacterial and eukaryotic cells. Upon light induction, two Vivid domains dimerize, bringing the N-terminus of one domain spatially close to the C-terminus of the other binding domain. The fusion of these domains helps in binding of the C-terminus of the N-terminal fragment to the N-terminus of the C-terminal fragment, reconstituting the enzyme in a spatial manner by incorporating optogenetic regulation into the T7RNAP through light-induced assembly and dissociation.



Fig.1 - T7RNAP is split at different positions and fused to light-inducible dimerization domains via linkers at the split site.

Properties of Opto-T7 system

1) Blue light-sensitive: pMag and nMag bind together only in the presence of blue light, which means the OPTO-T7 RNA Polymerase is active and transcribes only at a wavelength around 460 nm , thereby providing a light control for the production of the protein of interest.



Fig.2 - Illustration showing Opto-T7 RNA Polymerase .

2) Low dark state basal expression with a very high fold change in light-induced expression: With a reported fold change of greater than three hundred-folds (>300 folds) upon induction of light coupled with a very low dark-state expression (∼5-fold compared to the reporter control), the OPTO-T7 system is ideal for optogenetic control.


Fig.3 - A) Activation of Opto-T7 RNA Polymerase. B) Transcription of the gene of interest in the presence of blue light.

3) Fast reversal rate to the inactive dark state: We have used previously reported mutations in pMag (mutations I74V and I85V for pMagFast2) which has a dissociation time of 25 s in the absence of blue light. Therefore, the OPTO-T7 system can be used as an effective light-inducible expression system that reacts rapidly to changes in the light input.

Our constructs

Given below are the constructs we made for Opto-T7 RNAP System:



Fig.4 - pMag with GGSGG linker to C-Terminal of T7RNAP and GP2. (BBa_K3165052)



Fig.5 - pMag with GGSGG linker to C-Terminal of T7RNAP and mOrange. (BBa_K3165054)
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Fig.6 - nMag with GGSGG linker to N-Terminal of T7RNAP. (BBa_K3165050)

The T7 RNA Polymerase was split at the 563th/564th amino acid position because it was sterically suitable to attach the linkers at this position without affecting the activity of the polymerase. Also, this split was reported to have the highest fold change upon light induction. We've used a well-characterized 5 amino acid residue linker i.e. (Gly-Gly-Ser-Gly-Gly) to link pMag and nMag to the N and C terminal of the T7 RNA Polymerase.

All the sequences were codon-optimized for Bacillus subtilis. We had mOrange as a reporter for characterizing the system by shining light for different time durations and at different intensities at around 460 nm. In addition to this, we used a weak constitutive promoter (BBa_K823000) to avoid protein expression stress on the bacteria.

References:

(1) Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control Armin Baumschlager, Stephanie K. Aoki, and Mustafa Khammash ACS Synthetic Biology 2017 6 (11), 2157-2167 DOI: 10.1021/acssynbio.7b00169

CcdAB Antitoxin-Toxin System
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Introduction

Controller of Cell Division or Death B (CcdB) is the toxic component of the Escherichia coli CcdAB anti-toxin toxin system. It is a globular, dimeric protein with 101 residues per protomer, involved in the maintenance of F plasmid in cells by a mechanism involving its binding and the poisoning of Gyrase A subunit of DNA Gyrase, which leads to the breaking of the double-stranded DNA in the bacteria.

Cartoon working of CcdB toxin

Fig.1 Binding of CcdB toxin dimer to GyrA
In the presence of CcdA, CcdB forms a highly stable complex with it. Thus, the potency of the system is based on the relative concentration of the toxin to that of the antitoxin. Also, it has been found that the CcdB protein has a much higher half-life than CcdA. So, CcdB is a very potent bactericidal poison, and hence it cannot be used in growth control of bacterial populations (as intended by our project). It can be produced in the E. coli mutant Top10G R462C, which confers total immunity to the cell from the CcdB toxin. It cannot be expressed in most strains of E. coli, as even leaky transcription is enough to produce sufficient CcdB to kill the host. Hence, its use in other strains of E. coli is not possible.

Solution to this problem

We contacted various labs in our institute and found that Professor Raghavan Varadarajan, of the Molecular Biophysics Unit in IISc, works on various mutants of CcdB. There exist hyperactive and partially unstable forms of the protein, which could potentially be used in our project. Hence, we thought of using the CcdB L83S mutant, which is a highly unstable mutant of the wild type CcdB protein that has a mutation in the core region at the 83rd amino acid residue (which has been changed from leucine to serine). This change reduced the toxicity of the protein and has made it a viable option.

The ccdB L83S gene was placed under the araBAD promoter in order to produce the protein of interest. The CcdB system was incorporated in the Top10 pJAT strain of E. coli and leaky transcription was reduced by the addition of glucose as a repressor. pJAT plasmid confers homogeneity in the uptake of arabinose to the cells due to the constitutive expression of low-affinity high-capacity AraE Transporter.

Further, the CcdB L83S protein was purified using the CcdA column by the column chromatography technique. Thermal Shift Assay, Size Exclusion Chromatography and SDS PAGE Visualization were carried out for the characterization of the protein.

Our Construct

The construct used by us contains CcdB mutant L83S under an araBAD promer.

ccdB Construct

Fig.2 CcdB under araBAD promter (BBa_K3165048)

References

  1. Anusmita Sahoo, Shruti Khare, Sivasankar Devanarayanan, Pankaj C. Jain, and Raghavan Varadarajan "Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis" doi: 10.7554/eLife.09532
  2. Bharat V.Adkar, Arti Tripathi, Anusmita Sahoo, Kanika Bajaj, Devrishi Goswami, Purbani Chakrabarti, Mohit K. Swarnkar, Rajesh S.Gokhale, Raghavan Varadarajan "Protein Model Discrimination Using Mutational Sensitivity Derived from Deep Sequencing" https://doi.org/10.1016/j.str.2011.11.021
  3. Kanika Bajaj, Ghadiyaram Chakshusmathi, Kiran Bachhawat-Sikder, Avadhesha Surolia, and Raghavan Varadarajan "Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)"
  4. Artem Khlebnikov, Kirill A. Datsenko, Tove Skaug, Barry L. Wanner
    "Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter" and Jay D. Keasling1

Salient Features

  • We decided to use CcaR/S system for E. coli and not B. subtilis because CcaS is a membrane protein, and hence it can only perform its function in Gram-negative bacteria. Thus, the Opto-T7 system was assigned to B. subtilis. Another reason for the above choice is the high phototoxicity of blue light towards bacteria. This effect would be lower for bacteria with a thicker cell wall, hence the decision to induce Opto-T7 in a Gram-positive bacteria.

  • Further, we decided to use a combination of CcaSR v2.0 and v3.0 for a much higher dynamic range, which can theoretically also be controlled by modulating the intensity of the incident light. This also would ensure lower leakiness, and hence provide optimal growth.

  • We chose to use Gp2 as a bacteriostatic protein and not a bactericidal protein as it would be of much more benefit later on in industrial use/research use to arrest the cell growth rather than kill the cell for speedy control of bacterial population.

  • Similarly, we decided not to use CcdB L83S as the growth regulatory protein because it is a toxin system, and even though the mutant used by us is partially active and unstable, there is still a high risk of getting invalid results due to very recent emergence of CcdB L83S, and thus lack of information about its exact functioning.

  • Further it should be noted that CcdB cannot be used in B. subtilis, as it targets the GyrA subunit of gyrase in E. coli specifically, and its function in B. subtilis is unknown.