Team:Stockholm/Description

Project Abstract

Hypothesis

Status

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Biomarker recognition

Presence of certain proteins in blood or urine indicate disease. (REFERENCE). Most of these proteins are too large to pass through the membrane of bacteria without active transport. Thus, a bacterial assay to detect protein biomarkers must transport them into the cell or detect them with a receptor in the membrane. In this project, we wanted to construct a chimeric receptor for biomarker detection.

Background to the idea

An ideal system for detection of protein biomarkers is specific, sensitive, transferable and cheap. This means that an ideal receptor should bind to the target biomarker with high specificity and affinity. To make the system transferrable to many biomarkers, it should be possible to modify the receptor to change its affinity. Furthermore, binding should trigger an intracellular cascade that is suitable for a read-out system.

With these aims in mind, we were inspired by Chimeric antigen receptors (CARs), a proposed therapy for cancer. In their simplest form, CARs are fusions of single-chain variable fragments from monoclonal antibodies and the transmembrane and cytoplasmic parts of CD3 receptors. Treatment with CARs modifies T-cells from the patient to express receptors which are specific to a certain biomarker. These T-cells are then reintroduced into the patient to recognize and kill cancer cells. (REFERENCE). If a suitable scaffold receptor exists in bacteria it may be possible to develop a family of Bacterial antigen receptors (BARs). BARs would not be a treatment but at detection system for biomarkers in blood or urine samples.

In prokaryotes, two-component regulatory systems are a common mechanism for signal transduction through the membrane. Most often, a membrane-bound histidine kinase activates in response to changes in the environment. This activates response regulators by phosphorylation. One such system in Escherichia coli is the receptor EnvZ and its response regulator OmpR. The EnvZ/OmpR system regulates differential expression of the outer membrane porin proteins OmpF and OmpC.

EnvZ as scaffold for chimeric receptors

The function of the periplasmic region of EnvZ is poorly understood and its ligand is not known. However, the region responsible for signal transduction has been characterized and is located between the transmembrane helix and the histidine kinase domain. This region, known as the HAMP-domain, is present in many two-component regulatory system receptors. [REFERENCE]. EnvZ stood out as an interesting candidate for BAR since it has been used for chimeric receptors before. Most successful examples are chimeras of receptors that both have HAMP domains, but in at least one case this was not required for signal transduction. ^{LINK TO REFERENCE} In 2005, Christopher A. Voigt et al. made a light sensitive EnvZ fusion with the photoreceptor cph1 which does not contain a HAMP domain, proving that such chimeras are possible. ^{LINK TO REFERENCE}

A common strategy for chimeric receptors is to characterize many possible constructs. While it is difficult to find a functioning construct, recent research by Roger Draheim et al. has shown that receptors with the HAMP-domain can be tuned by moving aromatic residues next to the HAMP domain. By using this “aromatic tuning” it is sometimes possible to find a sweet spot where conformational change in the binding domain results in signal transduction. This recent research points towards EnvZ as a good candidate for chimeric receptors in synthetic biology.

Using Affibody Molecules to bind biomarkers

To use EnvZ to recognize protein biomarkers we had to find a suitable binding domain. Both the C and N terminals of EnvZ are in the cytoplasm, so a binding domain could not just be fused to the end of EnvZ. This meant that we needed to find a binding domain that was likely to be stable, fold well and bind even when fused to another protein on both ends. Antibody fragments and molecules that mimic antibodies are often used to bind to protein biomarkers. Many families of molecules fit this description. We chose to work with Affibody molecules, an antibody mimetic first described at KTH Royal Institute of Technology and further developed by Stockholm based company Affibody AB.

Designing the chimeric receptor

Affibody molecules are stable and easy to express in bacteria. The original Affibody scaffold is based on the Z-domain of Protein A in Staphylococcus aureus. Affibody molecules are only 58 amino acids long and structured as a three-helix bundle. By randomizing 13 amino acids in two of the helices, Affibody molecules with specific binding affinity can found. We chose to work with the Affibody molecule ZHER2:342 that binds HER2, a biomarker for certain aggressive types of breast cancer. The sequence of ZHER2:342 has been published and used in chimeric proteins by many independent researchers. [Reference ZHER2:342]. Affibody molecules with affinity for different proteins behave in a similar way upon binding [REFERENCE], so it is likely that a working system with ZHER2:342 can be adapted for other biomarkers.

To find a BAR that would activate upon binding we had to find a good location to insert the ZHER2:342 into EnvZ. Since EnvZ forms homodimers in the inner membrane [REF] and we wanted the function to be as similar to native EnvZ as possible, we needed to research it’s secondary structure. When expressed in the membrane, the N-terminus of EnvZ begins with 16 amino acids in the cytosol followed by a transmembrane helix. The following periplasmic domain is 123 amino acids long and largely uncharacterized. A second transmembrane helix then leads back into the cytosol to the signal transducing HAMP domain, followed by a histidine kinase.

Since the secondary structure of the periplasmic domain has not been determined experimentally, we had to rely on predictions. A first prediction was made using the RaptorX protein prediction server [CITATION]. We then consulted with researcher Roger Draheim who had run a prediction of the periplasmic domain with the Phyre2 tool [CITATION]. These predictions were very similar, and the figure below is based on the Phyre2 model and rendered with PyMol [CITATION]. Based on this prediction, Draheim suggested that the domain was structurally similar TIpB, another bacterial receptor found in H. pylori. In TIpB, two alpha-helices are responsible for dimerization. The rest of the periplasmic region in TIpB is composed of beta strands, coils and a single helix and forms a ligand binding domain. [CITATION Sweeney et al]. Based on this, we decided to insert ZHER2:342 into the [COLOR] region in [FIGURE]. This chimeric receptor is hereby referred to as EnvZ-Affibody chimera.

Four constructs of the chimeric receptor were designed. In the figure below, regions that were replaced with ZHER2:342 in each construct are colored red. [FIGURE and decriptive text].

Figure X: A and B: Periplasmic domain of EnvZ from amino acid 42 to 158. Regions replaced with Affibody colored red, where A corresponds to BAR 1 and BAR 2 and B corresponds to BAR 3. C: Affibody ZHER2:342.

Choosing the host

EnvZ is expressed in the inner membrane and activates OmpR in the cytosol. This presented a problem since the HER2 biomarker is to large to permeate the outer membrane of E. coli. Early in the project we believed that we could use only the epitope which the ZHER2:342 binds to. We later realized that this would not be possible since Affibody molecules do not bind denatured proteins.

INSERT DESCRIPTION ABOUT ROGERS STRAIN

We then chose between two alternatives. One was to express the system in a gram positive host. This would circumvent the problem since gram positive bacteria lack an outer membrane. This solution would be preferable and should be the aim for future applications of the system. However, since all previous work on EnvZ had been done in E. coli, getting the entire EnvZ-OmpR pathway to work in another host could well be a project of its own. Another option was to create spheroplasts by removing the outer membrane of the E. coli before adding the HER2 biomarker to the sample. We decided to go with the latter option of creating spheroplasts since this introduced fewer unknown variables when expressing the receptor in the membrane.

If spheroplasts are used to prove our concept, they must be able to express proteins to produce a read-out signal. Investigating this thus became a part of our project.

Stipulating hypotheses

Hypothesis 1: A construct of the EnvZ-Affibody chimera can be successfully expressed in EnvZ deficient E. coli
Hypothesis 2: The construct is expressed in the inner membrane
Hypothesis 3: A construct of the EnvZ-Affibody chimera protein binds the HER2 protein
Hypothesis 4: A construct of the EnvZ-Affibody chimera protein phosphorylates OmpR when it has bound the HER2 protein
Hypothesis 5: The read-out can be activated in spheroplast E. coli

...

Experimental plan

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Intracellular signal transduction

If activation of the histidine kinase on the BAR is successful, it will phosphorylate the response regulator OmpR. The aim then becomes to carry this intracellular signal from the receptor to the readout strain, together with quantification of the signal. The main idea is to build/assemble a BioBrick with OmpR dependent two quorum synthases that will produce two different quorum sensing molecules depending on the levels of phosphorylated OmpR inside the cell. Finally, these quorum sensing molecules are detected by the readout strain and quantified accordingly.

The whole signaling system is divided into two parts: i) Fluorescence signaling system (consists of Part A, B and C) to check if the BioBricks are functional and ii) Quorum sensing signaling system (Part D, E and F) to replace the fluorescence output with the quorum sensing output as final outcome.

Regulating protein expression with OmpR (Part A)

When EnvZ (histidine kinase) exhibits its kinase property, it leads to phosphorylation of OmpR which is an osmoregulatory protein in E. coli. Activation of EnvZ- OmpR two-component regulatory system is the main focus of this signaling pathway. Phosphorylated OmpR regulates differential transcription of two porin responsing genes (OmpC and OmpF)[REFERENCE] Activation of the BAR can either lead to kinase activity or phosphatase activity of EnvZ (depending on the conformational change in the receptor and the intracellular osmolarity) which in turn leads to dephosphorylation of OmpR inside the cell[REFERENCE] When the osmolarity is low, phosphatase activity of EnvZ is pronounced which leads to increased expression of OmpF gene and vice versa. Our first goal was to express RFP which is OmpR regulated.

[FIGURE]

Silencing protein expression with MicF RNA (Part B)

The micF gene has been shown to regulate post-transcriptional expression of OmpF gene. The micF gene encodes an antisense RNA which binds to its target region in OmpF gene, leading to inhibition of translation[REFERENCE]. Taken this fact into consideration, we tried to incorporate MicF Target (micF-T) and GFP in one plasmid in order to express GFP with a constitutive promoter.

[FIGURE]

Showing differential expression of RFP and GFP (Part C)

This is the final part of the fluorescence signaling system. In order to investigate both the effects of phosphorylated and dephosphorylated OmpR, this part is created. The main idea is to introduce micF RNA in a plasmid, together with Part A and Part B. In high intracellular osmolarity condition, micF binds to micF-T in Part B and silence production of GFP (Red colonies are pronounced here). In contrast, GFP is pronounced in low osmolarity when micF is not binding to micF-T. Thus, it makes the whole system sensitive to both kinase and phosphatase activity in the BAR.

[FIGURE]

Replacing RFP/GFP with quorum sensing molecules (Parts D&E)

After showing that differential expression regulated by OmpR is possible, we want to extend this system to production of quorum sensing molecules. Each fluorescent protein is replaced by a quorum synthase, keeping the backbone and other parts same. Part D is basically similar to Part A where RFP is replaced by RhlI synthase which produces OHL. In part E, GFP in part B is replaced with BHHL producing quorum synthase that is LuxI.

[FIGURE]

Putting it all together (Part F)

This is our final product in the signaling pathway which is a combination of part D, part E and micF RNA. The final goal of this project in the signaling section is to achieve a highly sensitive on-off switch mechanism in the OmpR dependent regulatory system depending on the kinase and the phosphatase activity of the BAR.

[FIGURE]

Stipulating hypotheses

Hypothesis 6: Expression of Part A leads to OmpR dependent production of red fluorescence protein (RFP).
Hypothesis 7: Expression of Part B leads to constitutive expression of green fluorescence protein (GFP) which can be silenced with MicF RNA.
Hypothesis 8: Expression of Part C leads to OmpR dependent regulation of RFP/GFP production.
Hypothesis 9: Expression of Part E leads to constitutive expression of quorum sensing molecule OHHL which can be silenced with MicF RNA.
Hypothesis 10: Expression of Part F leads to OmpR dependent regulation of BHL/OHHL production.

Experimental plan

Nunc vitae neque lorem. Interdum et malesuada fames ac ante ipsum primis in faucibus. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Duis vel ipsum quis sapien gravida maximus sit amet id ante. Vestibulum fringilla placerat neque eget laoreet. Nunc eget felis congue, tincidunt est in, egestas ipsum. Cras enim elit, elementum eget mattis id, tincidunt nec purus. Donec dignissim orci eget ante sagittis dapibus. Nam in laoreet sapien. Cras vitae ullamcorper dui, nec sollicitudin orci. Donec sagittis pharetra ex, eget ultrices nisl rhoncus eget.

Modeling the EnvZ/OmpR pathway

To predict the correlation between the concentration of activated EnvZ and signal intensity, we modeled the mechanism from EnvZ to OmpC and OmpF. Depending on the osmolarity level the output is either OmpC (in the case of high osmolarity) and OmpF (low osmolarity). By determining this connection we can then adjust the EnvZ amount to get the desired product, OmpC. After the OmpC translation, the two quorum sensing molecules (QSM), BHL and RhlR, are activated to further produce GFP. By analyzing the ratio at with OmpC triggers the QSM we can achieve a GFP production that is easy to detect. With this data we would, in theory, be able to link back to the EnvZ concentration. This means that a detectable level of the biomarker can be received.

The process by which we achieved these results was mostly researching the subjects and using Simbiology, an application in Matlab to both design and simulate models. For the third and fourth part of our project we started with the same methods as previously but realized early on that we needed to develop our own equation. For this we expanded our knowledge in Matlab and also contacted iGEM Technion from 2014 for support.

The hypothesis for the first part (EnvZ->OmpC/OmpF) was that EnvZ is proportional to the OmpR-P production but, at the same time, a part of the OmpR-P is consumed.