Team:HKUST/Proof Of Concept

ShellBi

Project
Description Design Engineering Proof of Concept Implementation Safety Contribution Notebook
Wet Lab
Experiments Results
Dry Lab
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Proof of Concept

Roadmap to Achieve ShellBi

The Concept

Our goal is to build a prokaryotic biosensor that gives a fluorescence readout on the detection of Paralytic Shellfish Toxins (PSTs). We exploit the antagonistic effect between PSTs and another drug called Veratridine (VTD) for this goal. PSTs are potent ion channel blockers while veratridine is a ion channel activator, i.e. it leads to extensive ion influx into the cell.

For the working of this biosensor, initially all cells are incubated with shellfish sample extract and later exposed to Veratridine. If the shellfish sample contains PSTs, ion channels would be blocked, leading to a decrease in osmolarity of the cell, despite the addition of veratridine. On the other hand, if toxins are not present, veratridine will cause an immense ion influx, increasing cellular osmolarity.

- Paralytic Shellfish Toxins
+ Veratridine

+ Paralytic Shellfish Toxins
+ Veratridine

Figure 1: The osmolarity and fluorescence of the cell in the absence or presence of PST

We are using the two component system (TCS) in E. coli to respond to the intracellular ionic changes. See below link for details of the circuit.

Design page

In order to prove that the biosensor works, we need to show:

• ❒ The toxins induce expected results on the chassis
• ❒ The fluorescence colours are expressed at appropriate osmolarity range
• ❒ The antisense RNA can indeed reduce the noise
• ❒ The scaffold and auto-inhibition can provide a robust control on the circuit

Toxins on the Chassis

Through a molecular docking software, Autodock vina, we were able to establish the binding properties of both veratridine (VTD) and saxitoxin (STX) on an important osmoregulatory ion channel of E. coli, MscS [1]. The software gives a score for ligand-receptor binding based on binding energies.

Evidence for STX binding:

• 1. We found that the score for STX binding to MscS, a mechanosensitive ion channel in E. coli, is similar to the score of STX binding to human Nav1.7 sodium channel. Scores for STX binding to MscS is -7.6 while for STX binding to Nav1.7 is -6.4
• 2. The STX molecule binds on the pore of the protein channel indicating that it could block the channel
• 3. We have evidence from literature that saxitoxin [2] and tetrodotoxin [3] block sodium influx into E. coli cells and affect their physiological characteristics

Evidence for VTD binding:

• 1. VTD binding energy to the MscS channel is similar to that of its binding with human sodium channels. Both scores are around -9
• 2. VTD molecule binds on the transmembrane region of the protein channel indicating that it modifies the channel’s ion gating properties
• 3. VTD binds to the channel with a higher binding energy than STX, which is in line with our literature review where VTD is speculated to have an irreversible binding on human sodium channels whereas STX binding is thought to be reversible

From this we know that the PST and Veratridine can induce the desired ion influx results in E. coli. Moreover, the two component system (TCS) is native to the organism, meaning that we can be certain, even as per our wet lab results, that the system will function well in E. coli.

Model page

Toxin Mimicked by Medium Osmolarity

Our project focuses on potent neurotoxins which require extensive care and precautions for handling. Besides the fact that our lab is not well equipped to handle such toxic chemicals, Hong Kong customs do not allow the entry of these toxins without special permits. For safety reasons, we are not going to use potent neurotoxins such as saxitoxin in our experiments. Instead, we mimic the conditions of the toxin’s presence by altering the osmolarity of the culture medium. We did so by adding NaCl to a final concentration of 0.033M, 0.048M, 0.078M, 0.113M, 0.171M, 0.214M, 0.256M, 0.299M.

The rationale behind varying osmolarity of the medium is that we know that the antagonism of toxins results in change of ionic levels in the cell, as seen in Figure 2. This ionic change impacts the intracellular osmolarity.

Figure 2: Change in ion influx upon addition of Veratridine 100μM (Na+ ▴ and K+ ▵) and Saxitoxin 1 μM (Na+ ▾ and K+ ▿)

We measure the impact the growth medium with increasing Na+ concentration has on the intracellular osmolarity. Using an osmometer, we found an exponential relationship between an increasing medium concentration and intracellular osmolarity. See below links for more information.

Experiments page

Results page

We draw a relation between intracellular osmolarity induced by the medium and the one published in literature using toxins to closely mimic the presence/absence of PSTs.

Figure 3: Linking of all concepts

Fluorescence Intensity Against Osmolarity Changes

When growing cells in media of increasing osmolarity, the intracellular osmolarity changes as well. We measure the fluorescence intensity of both green and red in all the different culture medium concentrations. We eventually plot the intensities of both the fluorescent colours against the intracellular osmolarities.
This gives a measure of which colour is more intense at each osmolarity.

Since the osmolarity variation here is a measure of whether toxin is present or absent, we wish to observe if red colour is indeed dominant over green at lower osmolarity, i.e. when toxin is present. Similarly, we expect green colour at higher osmolarity, i.e. when toxin is absent, indicating a clean sample.

The dry lab team has analysed the stability of this system and identified the most significant tunable parameters, which will allow us to optimise our circuit. The theoretical model of dry lab was also fit to the wet lab results to better prove that at a certain medium osmolarity, we did obtain the fluorescence colour of interest.

Figure 4: Fluorescence intensity trend of GFP and RFP with increasing intracellular osmolarity

Antisense RNA for Noise Reduction

We realise engineering 2 colours together in one chassis introduces noise and makes getting a discrete positive or negative output more difficult. Therefore, in order to suppress noise we wish to encode an antisense RNA against the undesirable fluorophore under the regulation of respective promoters. Since our fluorophores are regulated under different promoters, we express the antisense RNA against the fluorophore in its alternate promoter.

Figure 5: TCS modified by inserting mRFP1 and eGFP antisense under the OmpF promoter; while eGFP and mRFP1 antisense are controlled under OmpC promoter

In order to prove if the antisense RNA is effective in suppressing the signal from a fluorescent reporter, we developed a circuit with constitutively expressed fluorescence protein and compared its fluorescence intensity with when this construct was assembled with an antisense RNA against the protein. The antisense RNA was regulated under the promoter of interest (PompF or PompC) so this would allow us to also understand the activity of the promoter. Upon comparison of the two states- with and without antisense RNA, we observed that the fluorescence intensities from cells with an antisense RNA was significantly lower than when there was no antisense RNA present. This proves that our new circuit is effective in noise reduction.

Figure 6: Antisense RNA can significantly reduce expression of FP, hence, reducing noise

Scaffold, Autoinhibition and Robustness

Since our stakeholders and target users desire a fast, robust biosensor with high accuracy, we decided to engineer response regulators that favours one condition until a switch in system due to osmolarity change is detected.

Both response regulators of the Two Component System are engineered (see the link below) such that in low osmolarity their interaction will be suppressed until the protein scaffold is expressed which is only possible in high osmolarities..

Design page

At low osmolarities, the auto-inhibited response regulator will favour the production of RFP. The suppressed kinase activity of EnvZ will also allow stability of the signal. At high osmolarities, the scaffold will be expressed since it is regulated under OmpC, therefore fortifying the interaction and favouring the production of GFP. It can also create a feedback loop to speed up the response of the circuit to changing conditions.

To prove that this works, besides data from literature [4], we also wish to conduct our own experiments, by cloning the engineered response regulators and the scaffold protein under regulation of PompC. We will then compare the enhanced fluorescence intensities from the engineered TCS to the unmodified TCS circuitry.
This comparison would prove how effective the modular scaffold and auto-inhibition is in preventing cross talk, establishing stability and enhancing signal speed.

References:

1. Rasmussen, Tim. “How do mechanosensitive channels sense membrane tension?.” Biochemical Society transactions vol. 44,4 (2016): 1019-25. doi:10.1042/BST20160018

2. Tisa, L S et al. “Effects of organic antagonists of Ca(2+), Na(+), and K(+) on chemotaxis and motility of escherichia coli.” Journal of bacteriology vol. 182,17 (2000): 4856-61. doi:10.1128/JB.182.17.4856-4861.2000

3. Al-Balool, Fawzia Yaqoub. “Effects of tetrodotoxin and ion replacements on the short-circuit current induced by Escherichia coli heat stable enterotoxin across small intestine of the gerbil (Gerbillus cheesmani).” Memórias do Instituto Oswaldo Cruz vol. 99,2 (2004): 141-5. doi:10.1590/s0074-02762004000200005

4. 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, https://doi.org/10.1073/pnas.1209230109. Accessed 9 Oct 2021.

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