Project

How the BioPad works

Our self-designed PDMS microfluidic chip, the BioPad, is made of hundreds of compartments representing "pixels." Each 30µm x 30µm x 3µm compartment contains a few layers of E. coli. When the surface of the chip is touched, a deformation of the chip - and thus of the compartments - leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm. The aggregated/misfolded proteins are then sensed by the histidine kinase CpxA sensor, which auto-phosphorylates and transfers its phosphate to its corresponding relay protein, CpxR. Upon phosphorylation, CpxR homo-dimerizes. Our engineered bacteria contain CpxR proteins fused to split fluorescent or luminescent protein fragments (IFP1.4) via a 10-amino acid, 2x GGGGS flexible linker. Therefore, our engineered bacteria allow us to detect CpxR dimerization, synonymous periplasmic stress and touch. The split protein fragments are reversible. Therefore, when stress is removed, CpxA changes conformation and dephosphorylates CpxR allowing it to dissociate. The signal is shutdown and darkness returns The BioTouch Detector (composed of an inexpensive CMOS called Raspberry Pi, a highly sensitive digital camera with appropriate light filters, and a light emitting source) identifies and processes the position of the light/fluorescence emitted by the BioPad. This information about the position of the light relative to chip is then used to control the associated electronic device.

The CpxA-R Pathway

The natural function of the CpxA­-CpxR two component regulatory system in bacteria is to control the expression of ‘survival’ genes whose products act in the periplasm to maintain membrane integrity. This ensures continued bacterial growth even in environments with harmful extracytoplasmic stresses. The CpxA-R stress pathwayThe CpxA-­CpxR two component regulatory system belongs to the class I histidine kinases and includes three main proteins:

Engineering: CpxR and split complementation techniques

The main component that we wish to engineer is CpxR. It has been reported that the protein homo-dimerizes upon activation. We thus plan to use fused split proteins of fluorescent and bioluminescent nature to detect its activation.

table As a preliminary step, we used two fluorescent proteins sfGFP and IFP to characterisation CpxR. Split sfGFP (superfolder GFP) is an irreversible split system which will be used to prove the dimerization of CpxR. Split IFP on the other hand (Infrared Fluorescent Protein) is a reversible split system which will be used to understand the spatio-temporal dimerization of CpxR and thus allow us to better understand the On/Off mechanism of this system.

To achieve our final goal, we will engineer split bioluminescent proteins: Firefly and Renilla split Luciferases. These constructs will be the main component of our system. When fused to CpxR, we are expecting to witness emission upon touch.

Engineering: Microfluidic chip interface

Our specially designed microfluidic chip, hereafter known as BioPad Chip, will allow easy and accurate induction of fluorescent or bioluminescent signals. The chip is made up of thousands of compartments – representing the pixels of our device ­- of the height of a bacteria. The BioTouch Chip thus allows the effective trapping and induction of stress onto our engineered bacteria.

Engineering: The BioPad Detector

The signals induced by the BioTouch Chip are then processed by our self designed detection system: the BioTouch Detector. The BioTouch Detector is mainly made of a cheap computer (Raspberry Pi), a highly sensitive digital camera with appropriate light filters, and a light emitting source. The BioTouch Detector locates signals from various sources (infrared fluorescence, green fluorescence and luminescence), processes them and sends back the relative positions of the signals with respect to the BioTouch Pad. Thanks to this position, we are able to extract information such as giving a computer operating system that the position represents the position of the mouse on a screen, that the well at the given position is a suitable antibiotic candidate, or that a gene of interest has been activated. We therefore effectively control a computer or any other electronic device through a living interface: the BioTouch Pad.

YEAST

Upon a suggestion by Professor Maerkl, we decided to take a look at the High Osmolarity Glycerol(HOG) pathway in S. Cerevisiae and see whether we could expand our project into yeast cells. Similarly to the CpxR pathway, it is responsive to not only osmotic stress but also heat shock, nitrogen starvation and oxidative stress1. The HOG pathway is activated mainly by two membrane proteins Sho1 and Sln1, of which Sho1's role is thought to be monitoring turgor during cell growth2. From this we hypothesized that by tagging downstream proteins of the pathway, PBS2 and HOG1, with split constructs we would be able to create a touch responsive system. PBS2 and HOG1 are part of a Mitogen-activated Protein Kinase(MapK) pathway, and PBS2 is a MAPKK and HOG1 is a MAPK so PBS2 phosphorylates HOG1 upon activation. PBS2 Map kinase kinase of the HOG pathway localized in the cytoplasm and with 2160 molecules/cell3. Typical MapKK structure with C-terminal protein kinase domain and N-terminal upstream protein interacting domain. HOG1 Map kinase of the HOG pathway localized in the cytoplasm and with 6780 molecules/cell3. Typical MapK composed only of a protein kinase domain which localizes to the nucleus upon activation.

But why yeast?

In synthetic biology, the most used organism is undoubtedly E.Coli. The easy nature of its manipulation makes it ideal especially when the time for the project is limited to a few months. However, studies in bacteria are not always applicable to all cells, and an extension to a eukaryotic species adds another dimension to our project. Especially in the case of the HOG pathway, there is a very similar pair of proteins in mammalian cells, MEK5 and ERK5, meaning that the project could be extended even further4. Using S. Cerevisiae and homologous recombination, we could also address other problems. In bacteria, inserting plasmids can disrupt the equilibrium of the pathway and activate it or deactivate it constitutively because the copy number will be changed. Also the project consisted in attaching a split construct to a homodimerising protein so there would be many interactions which would not produce the signal. However, there weren't only advantages in choosing to work with yeast. First, we had no idea how to work with yeast cells and neither did our assistants; we had to do a lot of research on basic protocols. Also, we could not find a study which had already tested the interaction between two proteins in the pathway using split constructs so we were making a small gamble. And last but not least, working with yeasts takes a lot longer due to the slower doubling time.

Why PBS2 and HOG1?

As mentioned above, we were unable to find a set of proteins which had already been assayed by split complementation. This meant that we would not be sure whether the split constructs would be able to interact once fused to the proteins and which orientation to use. We chose PBS2 because this is where the stress signal converges in the pathway from different membrane sensory proteins. And we chose HOG1 as the other protein because it is phosphorylated directly by PBS2 while there is a scaffold for the upstream interactions. For the orientation, there was no doubt that we had to use the C-terminal for PBS2 because the kinase domain interacting with HOG1 is on the C-terminal and the N-terminus is physically very far. We would have liked to test both orientations on HOG1 but due to limitation of time and people to invest on this part, we decided on the C-terminus. This choice was backed by the fact kinases have their regulation tail on the N-terminus and we did not want to risk disrupting its activation.

Learning how to use yeast

Having had several courses where yeast had been mentioned, we initially thought that we would not have too much trouble understanding how to take on the project. Little did we realise how much we were wrong. It took a whole month to fully understand what we had to do in terms of the design of the gene insert and order the plasmids we needed while we prepared everything we needed. Not knowing that the number of base pairs needed for homologous recombination varies between different species of yeast confused us, and then we were confused whether we should transform with a plasmid or linear fragments. In the end, we found plasmids specifically designed for GFP-tagging in yeast on AddGene and we consulted the professor Simanis and his assistant Andrea Krapp who helped us confirm our design.

How did we make our DNA constructs and transform our cells?

After the long discussion mentioned above, the plasmids we found were the plasmids pFA6a-link-yoSuperfolderGFP-(Kan and Ura) which are for GFP tagging via homologous recombination in yeast.

We replaced the GFP tag by using overlap PCR and created split Renilla Luciferase, split GFP and whole Renilla Luciferase tags to finally produce linear fragments of the form below. The figure illustrates how the modified Genome would look like and what proteins it would express.

It took us many PCRs to produce the 8 different linear fragments we wished to use and after a failed transformation, we hypothesized that it was due to the bad quality of the products and took our time to optimize the reaction. We preferred to do subsequent transformations but due to a lack of time we also ended up trying the co-transformation while waiting for the verification of the first successful transformations.

Experimentation on the cells

NOTHING FOR THE MOMENT

References

1. The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccaromyces cerevisiae CTT1 gene; Shuller and al (EMBO journal, 1994) 2. Osmotic Stress Signaling and Osmoadaptation in Yeasts; Stefan Hohmann (Microbiol Mo Biol Rev. 2002) 3. Yeast GFP library; Ghaemmaghami S, et al. (2003) 4. Scaffold proteins of MAP-kinase modules; D N Dhanasekaran et al (2007)