Team:HKUST/Design

ShellBi

Project
Description Design Engineering Proof of Concept Implementation Safety Contribution Notebook
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Design

Building of Circuit

The Concept

Shellfish toxins like saxitoxin, tetrodotoxin, brevetoxin, etc. are known to block human Nav1.7 sodium channels, thus they are paralytic and neurotoxic in nature- called Paralytic Shellfish Toxins (PST). However, ion channels in prokaryotes do not respond to PSTs the way eukaryotes do. Hence, if bacteria or cyanobacteria (Pomati et al., 2004) are treated solely with the toxin, there is no significant observable cellular change.

Normal Cell Condition

Addition of Veratridine

Addition of Saxitoxin + Veratridine

Table 1: Veratridine and saxitoxin antagonism in cyanobacteria

Veratridine, on the other hand, is an antagonist to such toxins. It is relatively less toxic, can bind to the ion channels of prokaryotes and induce a massive ionic influx into the cell which eventually leads to cell lysis. When both Veratridine and Saxitoxin are added prokaryotic cells, it has been reported by Pomati et al. (2004) that the channel blocking effects of the toxin become more apparent[1]. As seen in Table 1, the sodium and potassium ion levels see a surge upon the addition of veratridine. This is contrasted by a drastic decline in ionic concentrations upon the addition of saxitoxin. The overall change in cellular ionic concentration is a direct indicator of the presence of PSTs.

As the ionic levels contribute to the changing osmolarity within the cell, we want to create a genetic mechanism that responds to this osmolarity change as an indicator of the presence of toxins. The Two Component System enables us to do so...

Two Component System (TCS)

Figure 1: Native two component system in Escherichia coli

The two component regulatory system, composing the EnvZ/OmpR is studied vastly in E. coli. EnvZ/OmpR are cognate response regulators that, when under the stimulus of an osmotic stress, modulate gene expression of two major outer membrane porins: OmpC and OmpF (Figure 1). OmpC has a smaller pore diameter and is expressed under hyper-osmotic conditions while OmpF, having a wider pore size, is expressed under hypo-osmotic conditions [2]. The difference in channel sizes allows for a more regulated rate of diffusion of small molecules essential for survival.

EnvZ is a histidine kinase receptor-like protein in the inner membrane that can undergo auto-phosphorylation under increased osmolarity in the cell. The phosphorylation of this protein leads to its kinase activity on its cognate response molecule, OmpR. Phosphorylated OmpR (OmpR~P) will then form a dimer complex that can act as a transcription factor for the genes OMPF and OMPC [2].

Amount of OmpR~P determines whether OmpF or OmpC gene is activated

Figure 2: Affinity binding model of OmpF and OmpC promoter

Under high osmolarity, the kinase activity of EnvZ dominates, creating more OmpR~P. On the other hand, under hypoosmotic conditions, EnvZ could act as a phosphatase, removing the phosphate group from OmpR~P [3]. The relative difference in the amount of OmpR and OmpR~P in the cell is the main sensor that modulates gene expression.

A simple affinity binding model explains the regulation of the two OmpF and OmpC genes (Figure 2). High affinity binding sites can bind OmpR at even low concentration but low affinity binding sites need higher concentration of OmpR [4].

At low concentrations of OmpR~P, initially the higher affinity binding sites- F1, F2, and F3 on the OmpF promoter (PompF) are occupied which positively regulate and activate the gene. On the other hand, with increasing concentration of OmpR~P, the lower binding affinity regions namely C1, C2, C3 on the OmpC promoter (PompC) and the negative regulator of the PompF F4 is occupied. This results in the preferential expression of OmpC while suppression of OmpF porin. (Fig. 2)
We placed fluorescent proteins under the regulation of the OmpF and OmpC promoters (Fig 3):

• 1. Red Fluorescence Protein (mRFP1) with OmpF promoter,
• 2. Green Fluorescence Protein (eGFP) with OmpC promoter.

We expect to see green under high osmolarity and red under low osmolarity conditions.

Figure 3: TCS circuit engineered with mRFP1 under PompF and eGFP under PompC

Through the engineering cycle, we figured certain modifications that are required for the green reporter and the PompF promoter. See Engineering for details.

Engineering page

Antisense RNA

We realize the issue of leaky promoter expression of at least PompC (as noted by SDU Denmark as well as University of Indonesia iGEM teams) [5] and how this negatively affects the fluorescence readout. Moreover, we needed to ensure that customers see discrete ‘red’ or ‘green’ as results of the biosensor. In order to better control the noise, we encoded antisense RNA against the fluorescence proteins under alternative promoters. The native Two Component System had a major influence on this engineering decision. The OmpC promoter encodes a region annotated as “MicF” which serves as an antisense RNA to the OmpF porin mRNA [6]. Since we eliminated the porin gene, we did not include this segment in our circuit.

Figure 4: TCS modified by inserting mRFP1 and eGFP antisense under the OmpF promoter; while eGFP and mRFP1 antisense are controlled under OmpC promoter

The antisense RNA against mRFP1 is encoded under the OmpC promoter while the antisense for eGFP molecule is encoded under the OmpF promoter. So, at any given intracellular osmolarity only one colour protein of interest will be abundant, alongside the antisense molecule of the other fluorescent protein. The antisense fragment is 24 nucleotides long with 10 nucleotides annealing on the promoter -10 region which blocks the RBS of the target protein. Furthermore, they are also qualified with an Hfq-binding domain

Engineering Response Regulators
+ Scaffold Mechanism

In order to provide stability to the circuit as well as establish a feedback loop to speed up the reaction time of the circuit, we have engineered the EnvZ and OmpR as well as encoded a scaffold that could modulate the interaction between these two response regulators.

There are two features to this engineering segment:

Scaffold modulated response ❒ Enhances interaction between EnvZ and OmpR to upregulate kinase activity. Encoded only under PompC

Auto-inhibition of EnvZ kinase activity ❒ Prevents kinase activity of EnvZ while the PompC is not active, in order to favour and sustain PompF activity

Scaffold

Figure 5: Scaffold expressed under PompC of the basal TCs circuit

The EnvZ is engineered to have a SH3 domain of peptide sequence:

attached by a linker containing a 12 aa glycine/serine peptide chain on its C-terminus.

Figure 6: Engineered EnvZ with SH3 binding domain

Meanwhile, OmpR is engineered to have a leucine zipper adapter with sequence:

• SVKELEDKNEELLSELYHLKNEVARLKKLVGER

on it’s C terminus separated by a similar G/S linker sequence.

Figure 7: Engineered OmpR with a Leucine zipper binding domain

The interaction between EnvZ and OmpR is completed with the addition of a scaffold. The scaffold contains a Leucine Zipper binding domain

• SVKELEDKNEELLSRIYHERNEVARLKKLVGER

on one end, and an SH3 ligand

• PPPVPPRRRR

on the other end separated by a linker chain. This scaffold’s SH3 ligand binds the SH3 domain on EnvZ while the Leucine Zipper end of the scaffold forms a dimer with the leucine zipper adapter on the OmpR.

Figure 8: Engineered scaffold with Leucine Zipper ligand and Sh3 ligand

The choice of SH3 domain-ligand and Leucine zipper pairs is based on their successful use in several previous synthetic biology studies [7, 8, 9, 10].
The fortified interaction between the two components of the two component system increases the local concentration of proteins, and enhances phosphotransfer between response regulators. This would result in a more rapid response within the cell. The scaffold is only encoded under the PompC. Read the auto-inhibition attribute on EnvZ to learn how the circuit is completed.

Figure 9: Complete engineered TCS response regulators

Auto-inhibition of EnvZ

The auto-inhibition attribute of EnvZ functions to prevent the kinase activity of EnvZ in low intracellular osmolarity. Thus, maintaining the low copy of OmpR~P in low intracellular osmolarity and only PompF will be expressed.

Figure 10A: Swiss-Model of WT EnvZ cytoplasmic domain

Figure 10B: Swiss-Model of EnvZ cytoplasmic domain with auto-inhibitory SH3 ligand inserted

The auto-inhibition is achieved by engineering the EnvZ to have an SH3 ligand in the middle of the protein. The SH3 ligand peptide is inserted near the turn between the helices of the important phosphotransfer domain of EnvZ. The goal is that the SH3 domain on the C terminus of protein could bind to this internal SH3 ligand (Kd 5uM) and it would prevent phosphotransfer to OmpR as long as the scaffold is not involved. Since the scaffold has an SH3 ligand with a smaller dissociation constant (Kd 0.1uM), the auto-inhibition will be released and kinase activity will be resumed with its addition.

Figure 11A: Kinase activity of EnvZ is activated since auto-inhibition is lifted

Figure 11B: EnvZ auto-inhibition causes the kinase activity of EnvZ to be disrupted

The scaffold is encoded under the OmpC promoter such that it will be expressed under high intracellular osmolarity. As a result, in lower osmolarity, the phosphotransfer between the response regulators is restricted and PompF will be able to have a stable expression while the PompC activation will be inhibited.

In higher osmolarity, the scaffold will bind to OmpR and compete with the auto-inhibitory SH3 ligand. Since the dissociation constant for SH3 ligand on scaffold has a lower Kd, it will preferably bind to the SH3 domain. The auto-inhibition will then be obstructed, while the kinase activity of EnvZ on OmpR will be enhanced. The high concentration of OmpR~P will eventually form a positive feedback loop for the activation of PompC leading to a sharper, and a rapid signal.

Our circuit at the moment assumes that the PompC has enough leaky expression to produce small amounts of scaffold in order to facilitate the production of OmpR~P required for PompF activation. Alternatively, since we did not knock out the native TCS in E. coli, we rely on the native presence of OmpR~P for a proof of concept regarding our engineering.

The table below is a summary of how the circuit would respond to the PSTs:

Absence of PST

Presence of PST

References:

1. Pomati, Francesco et al. “Effects of saxitoxin (STX) and veratridine on bacterial Na+ -K+ fluxes: a prokaryote-based STX bioassay.” Applied and environmental microbiology vol. 69,12 (2003): 7371-6. doi:10.1128/AEM.69.12.7371-7376.2003

2. Kenney, Linda J, and Ganesh S Anand. “EnvZ/OmpR Two-Component Signaling: An Archetype System That Can Function Noncanonically.” EcoSal Plus vol. 9,1 (2020): 10.1128/ecosalplus.ESP-0001-2019. doi:10.1128/ecosalplus.ESP-0001-2019

3. Zhu, Y et al. “Phosphatase activity of histidine kinase EnvZ without kinase catalytic domain.” Proceedings of the National Academy of Sciences of the United States of America vol. 97,14 (2000): 7808-13. doi:10.1073/pnas.97.14.7808

4. Head, C G et al. “Relative binding affinities of OmpR and OmpR-phosphate at the ompF and ompC regulatory sites.” Journal of molecular biology vol. 281,5 (1998): 857-70. doi:10.1006/jmbi.1998.1985

5. "Part:Bba R0082 - Parts.Igem.Org". Parts.Igem.Org, 2021.

6. Delihas, N, and S Forst. “MicF: an antisense RNA gene involved in response of Escherichia coli to global stress factors.” Journal of molecular biology vol. 313,1 (2001): 1-12. doi:10.1006/jmbi.2001.5029

7. Posern, Guido et al. "Development Of Highly Selective SH3 Binding Peptides For Crk And CRKL Which Disrupt Crk-Complexes With DOCK180, Sos And C3G". Oncogene, vol 16, no. 15, 1998, pp. 1903-1912. Springer Science And Business Media LLC.

8. Dueber, John E. et al. "Reprogramming Control Of An Allosteric Signaling Switch Through Modular Recombination". Science, vol 301, no. 5641, 2003, pp. 1904-1908. American Association For The Advancement Of Science (AAAS),

9. Dueber, John E et al. "Engineering Synthetic Signaling Proteins With Ultrasensitive Input/Output Control". Nature Biotechnology, vol 25, no. 6, 2007, pp. 660-662. Springer Science And Business Media LLC.

10.Whitaker, W. R. et al. "Engineering Robust Control Of Two-Component System Phosphotransfer Using Modular Scaffolds". Proceedings Of The National Academy Of Sciences, vol 109, no. 44, 2012, pp. 18090-18095. Proceedings Of The National Academy Of Sciences.

PROJECT

• Description
• Design
• Engineering
• Proof of Concept
• Implementation
• Safety
• Contribution
• Notebook

WETLAB

• Experiments
• Results

DRYLAB

• Model

REACH

• Human Practice
• Collaboration
• Communication
• Education
• Sustainable

PARTS

• Basic Parts
• Composite Parts

TEAM

• Team
• Attributions