Team:BGU Israel/test/Collaborations


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Team:BGU Israel



Collaboration


We have collaborated with a couple of iGEM teams this year:



Introduction


Stockholm team is working on cancer diagnosis by engineering of a bacterial sensor which could detect trace amounts of cancer biomarkers.


Stockholm Abstract:

"Synthetic biology offers many potential cost-effective healthcare technologies. One of those could be new ways to diagnose and treat disease at an early stage. Current techniques for biomarker detection (e.g. ELISA, RIA) are time consuming, expensive and require specialised equipment.

We intend to design a microbiological system for the detection of low quantities of biomarkers. This assay aims to be easier and more cost efficient than existing techniques and possible to perform in modestly equipped settings. Initially, we will focus on the expression of a receptor for the desired biomarker. Depending on the nature of the biomarker, the receptor will be either be native or designed.

Upon biomarker detection, signal amplification will be triggered by our receptor system to activate a read out/detection system (e.g. Luciferase, GFP) inside the microorganism to artificially amplify the extracellular signal. In the next stage, the team will go on to design a read-out system to measure the concentration of biomarkers in body samples. Finally we want to investigate if we can make this system transferable to other biomarkers, changing only the receptor part of the system.

The system would be cheap, fast and possible to distribute without advanced equipment. Our motivation is to improve patient prognosis and quality of life and to improve efficiency and reduce costs within the healthcare system."


Our assistance to the team is to express an GPI-anchored Affibody [biobrick number??+link] on the plasma membrane of human cancer cells, which can then be specifically targeted by a compatible drug or toxin (Fig. 1).



Fig. 1. Possible applications of Affibody expression in cancer cells.

Results


1. Cloning

We cloned the Affibody [biobrick number??+link] into our AAV vector for expression under CMV promoter (Fig. 2).

A
B

Fig. 2. A. Cloning map of Affibody into AAV vector. B. Successful cloning results.
2. Affibody expression in human cancer cells

Affibody-AAV was produced [link to protocol of virus production], and used for transduction of human cancer cell line (HT1080 fibrosarcoma cell line].
As the Affibody was predesigned to include 3xFLAG peptide (DYKDDDDK) in its sequence, we used anti-FLAG-specific primary antibody to specifically detect the protein using immunofluorescence.

Results

Conclusion and future aspects

Affibody construct could be successfully expressed in human cancer cells on cell membrane. In the future, the Affibody could be expressed exclusively in cancer cells using our Boomerang approach using dCas9-VP64-gRNA system under the control of cancer-specific promoters.




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