Team:HKUST/Contribution

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Contribution

Our Contribution to the IGEM Society

Overview

The EnvZ/OmpR two-component system (TCS) from E. coli is an essential part of our project. The TCS is in fact a very attractive target for synthetic biologists since it is a modular signalling pathway in prokaryotes, allowing genetic engineers to intervene at multiple parts of the pathway. The most common way to exploit this signalling pathway is to fuse the Histidine Kinase (EnvZ) with other sensory proteins that respond to induced effects, and then observe the effect-induced activity of the OmpC promoter.

Classic example is University of Indonesia, 2019 iGEM project where they had fused heparin-binding epidermal growth factor-like growth factor (HB-EGF) with the kinase domain of EnvZ [1]. The binding of sensor protein relayed a signal through the TCS and produced a GFP reporter under regulation of the OmpC promoter. Other uses could include fusing of Taz protein with EnvZ kinase domain such that the TCS is induced by addition of aspartate [2].

Having worked extensively with the EnvZ/OmpR Two Component System this year, we contribute to the iGEM society by providing:

• ❒ Characterisation of OmpC promoter response to increasing osmolarity
• ❒ Enhancement of Protein-Protein Interaction and prevention of cross-talk in signalling pathways using modular scaffold
• ❒ Improved model of EnvZ/OmpR TCS for future dry lab simulation and modelling
• ❒ E. coli potentially responding to PSTs allowing its applicability in biosensors
• ❒ Significantly improving a part, BBa_R0084- Characterisation of the negative regulatory region (F4) on OmpF promoter

Characterisation of OmpC promoter

This promoter is activated by a transcription factor OmpR which is the response regulator of the Histidine Kinase. We, here, provide characterisation of the OmpC promoter. In our case, we observed the TCS when cells are exposed to a medium with increasing osmolarity.

OmpC promoter (BBa_R0082) regulated eGFP

Figure 1: OmpC regulated eGFP increasing in intensity with increasing medium concentration

With increasing medium osmolarity, as a result of increasing medium Na+ concentration, the eGFP intensity is seen to increase 2 fold. The intensity increases from 10,000 AU to 20,000 AU. The samples were excited at 488 nm and emission was read at 507 nm. A negative plasmid control with WT BL21 cells is also measured for fluorescence intensity. Therefore, we can conclude that PompC is favourably activated at higher osmolarities.

We also conducted another experiment with the OmpC promoter regulating an antisense RNA against constitutively expressed mRFP1. For a control, we observed the fluorescence intensity of just constitutively expressed mRFP1 (Figure 2).
The difference in the intensities of the construct with only Constitutively expressed mRFP1 (BBa_J23100, BBa_E1010) and the construct with Constitutively expressed mRFP1 with PompC regulated antisense RNA was recorded to assess the behaviour of OmpC promoter (Figure 3). The rationale here is that at higher osmolarity of the medium, the OmpC promoter would be more active, therefore the fluorescence intensity from the cell construct would be much lower than its constitutively expressed fluorophore counterpart.

As seen in the graph below, the PompC regulated antisense RNA indeed is able to suppress fluorescence at higher medium osmolarities. This supports the hypothesis since the antisense RNA is under the regulation of the OmpC promoter, which is more active at higher osmolarities. However, surprisingly, the efficiency of the promoter or perhaps even the precision of measurement seemed to drop at higher osmolarities. It could either be that the cell was undergoing extreme stress at such high osmolarities and the death rate was higher, which renders the data beyond a 0.256M of NaCl in LB inconclusive.

Figure 2: Comparison of fluorescence intensity from constitutively expressed mRFP1 and PompC regulated antisense RNA against the fluorophore

Figure 3: Difference in intensities from constructs with and without antisense RNA as a measure of promoter function

Scaffold kit for enhancing Protein-Protein Interaction (PPI)

Scaffolds are proteins that function to bring proteins close together and stabilise their configuration. The functions and advantages of scaffold proteins are:

• • Stabilising and Enhancing interaction of proteins
• • Speeding up downstream signalling
• • Preventing cross-talk of signalling pathways
• • Allows the tuning of Protein-Protein Interaction

Phosphosignalling pathways, like the EnvZ/OmpR Two Component System (TCS) that we used, are attractive options for synthetic biologists as they offer a modular system to build on. Our scaffold mechanism is especially useful if two interacting proteins native to one organism are expressed in another chassis because often the host’s native signalling pathway interferes and cross-talks with the transformed components [3]. With the expression of specially engineered components and the scaffold we describe, it is possible to achieve a more specific downstream signalling cascade.

We worked with the Two Component System (TCS) which is a predominant modular signalling pathway in prokaryotes. We incorporated a scaffold mechanism to enhance the rate of transcription of promoter OmpC regulated eGFP reporter. Although we used this engineered scaffold system on our response regulators to enhance the phosphorylation rate, our system could be generalised to other interacting proteins as well.

Design

With reference to Figure 4, Protein A is modulated with an SH3 ligand on its C terminus, while Protein B is modulated with a Leucine Zipper binding ligand, also on the C terminus. Both the additional components are linked to the desired protein with an inert 12aa Glycine/Serine chain. The scaffold is designed to have one SH3 binding domain and one Leucine Zipper binding domain, with a similar linker chain for both modifications. The interaction of the proteins is fortified once the scaffold is expressed, which binds both the proteins.

Figure 4: Diagram of enhanced Protein-Protein Interaction with engineered modular domains and scaffold

How To Use

Both the engineered components, the SH3 ligand and Leucine Zipper binding ligand, are present on the C terminal of interacting proteins. We have prepared a ‘gene kit’ where future teams could insert their protein CDS between the engineered components.

The assembled gene circuit for enhancing PPIs should look like Figure 5 below. The desired protein’s CDS should be modified with SH3 domain (BBa_K4061110). The stop codon is provided after the domain, therefore the protein CDS must not contain the stop codon. Another interacting protein follows the same rules and is modified with a Leucine Zipper domain (BBa_K4061115).

The scaffold protein remains standard with the given domains. It can be expressed under any desired promoter.

Figure 5: Assembly and usage of the scaffold gene kit to enhance protein-protein interaction

Device BBa_K4061200 compiles all the components for this modular mechanism of protein-protein interaction enhancement.

Testing the Efficiency

In order to better characterise the working of this scaffold, teams are encouraged to test two interacting protein partners, perhaps even EnvZ and OmpR as provided by our team, in the engineered and unengineered form. We faced challenges obtaining a clone for this construct which led to delay in obtaining data, however, we encourage other teams to try this molecular scaffold and characterise it further. According to Whitaker, W. R (2012) the response of the non-cognate response molecule of EnvZ, CusR and CpxR, was greatly enhanced upon the addition of this molecular scaffold. To characterise the working of this scaffold dependent engineered system, we are required to test this modular scaffold using other proteins and compare their interaction with and without the engineered system.

We are in the process of cloning:

• 1. Constitutively expressed engineered EnvZ and engineered OmpR- scaffold expressed under OmpC promoter
• 2. Constitutively expressed engineered EnvZ, OmpR and constitutively expressed scaffold
• 3. Constitutively expressed non-engineered EnvZ and OmpR.

In 1, we could potentially create a positive feedback loop for OmpC activity, while 2 and 3 are for establishing control over both OmpC and OmpF promoters of the TCS.

Modeling of the EnvZ/OmpR Two Component System

In our dry lab modeling team, when we were constructing the model for the TCS, we found that different studies propose different models for the molecular mechanism of the signalling pathway. For the transcription factor OmpR, there are various models with different orders of phosphorylation, dimerisation, and DNA binding. We have done a comprehensive literature review to find the most probable model that is backed up with sound evidence and also compatible with the results from different sources.

We propose a model for PompC and PompF expression that combines the effect of

• • Binding of OmpR~P as a dimer
• • The reciprocal expressions of PompC and PompF
• • The hierarchical and cooperative binding sites on the promoters

Besides, previous studies on the TCS model only tackle a part of the system, i.e. the EnvZ-OmpR system and the PompC/PompF expression. In our model, we link the two models, with the consideration of the OmpR functioning as a dimer. Additionally, we have successfully fit the wet lab results to our model. See modelling.

Model page

E. coli sensitive to Saxitoxin and Veratridine Antagonism

To search for evidence of the response of E coli to Paralytic Shellfish Toxins and veratridine(VTD), we have carried out molecular docking on an E. coli ion channel MscS. We found that it has a similar binding affinity to Nav1.7, a human voltage-gated sodium channel that is known for causing Paralytic Shellfish Poisoning induced by saxitoxin(STX). Our findings also support the hypothesis that VTD irreversibly affects the gating of the channel while STX might have a stronger binding with the channel. See Proof of Concept for details.

Proof of Concept page

Significantly improving a part- OmpF promoter

Since our biosensor is based on the concept of measuring intracellular osmolarity, we chose to use a promoter that responds to low osmolarity in the E. coli cells - OmpF promoter. We found a part in the registry BBa_R0084 and although this part is the OmpF promoter, it contains only the positive regulators. We realised there was a negative regulator region of this promoter, therefore we obtained the OmpF promoter sequence from the E. coli K12 strain genome directly. We matched our sequence with the ones presented in studies [4].

We wished to test the effect the negative regulator region, annotated as F4, has on the promoter activity.
We synthesised the full OmpF promoter with the negative regulator region (-360 to -380bp) via IDT. This was ligated with the mRFP1 reporter. Through PCR, we obtained the sequence of the OmpF promoter without the negative regulator, 108bp long. This was also measured through the mRFP1 reporter.
We characterised the promoter activity via fluorescence spectrophotometer in a 96 well plate. Negative control of untransformed WT BL21 cells was used.

Figure 6A: Old part BBa_R0084 characterisation with an increasing osmolarity medium

Figure 6B: New part BBa_K4061000 characterisation with an increasing osmolarity medium

The new part (BBa_K4061000) that contains the F4 region has a steeper and a greater decline in fluorescence intensity at higher osmolarities. While the BBa_R0084 promoter reduces the fluorescence intensity only 1.6 folds, the construct with the negative regulator, BBa_K4061000 reduces almost 2 fold. Moreover, the new part, at higher osmolarities reduces to almost wild-type cell level, indicating the promoter is in fact ‘turned off’ at high osmolarity. The promoter without F4 has a higher fluorescence intensity, however, even at high osmolarities, the expression of the reporter seems to be significantly high to indicate that the promoter is not successfully regulated at non-favourable conditions (here high osmolarity). From this we can conclude that the F4 region on the promoter makes the promoter more sensitive to an increasing osmolarity also allowing a discrete ‘switch’ like behaviour between OmpF and OmpC promoters.

References:

1. “Team:UI Indonesia/Design - 2019.Igem.Org". 2019.Igem.org, 2021.

2. 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.

3. Pawelczyk S, Scott KA, Hamer R, Blades G, Deane CM, Wadhams GH (2012) Predicting Inter-Species Cross-Talk in Two-Component Signalling Systems. PLoS ONE 7(5): e37737.

PROJECT

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