Alpha System

The Alpha System is the preliminary design we planned for a biosensor that can detect various materials at low concentrations.
The main goal of our design is to make the bio-sensor capable of detecting harmful substances at far lower concentrations than existing bio-sensors. To do this, we designed a system which produces a chain reaction resulting from the input of a single molecule. This system utilizes a Cell-to-cell communication channel called quorum sensing, to make it possible for a cell which sensed a single molecule of the material detected to “inform” the surrounding cells of the existence of this toxin, causing them to produce a signaling molecule, and then inform their neighbors and so on, creating a chain reaction.
Unlike other biosensors, our system is meant to be as versatile as possible- with a minor change at the genetic level it can detect molecule A instead of molecule B. That saves us the need to construct an entirely different detector for every dangerous material out there.

The alpha system consists of three gates.
Each gate plays an important role in the detection process inside the cell, but the system as a whole is also based on communication between bacteria via quorum sensing.

Gate 1

Built and Biobricked (BBa_K1343000)

The goal of the first gate is to produce the quorum sensing molecule, AHL, when a material of interest is present in the bacterias' environment. As a result, this part can be replaced by any bio-sensor which uses AHL production as its output.

The construct includes a changeable promoter for the detection of the material of interest. In the example above the promoter is Ptet which is repressed by tetR; when tetracycline or its analog aTc is present, tetR is removed
When the material is present it will get into the cell, attach to the promoter and the transcription of the LuxI gene will begin. LuxI gene encodes for an AHL synthase. AHL is a signal molecule that can diffuse freely into the cell and out.
The NheI restriction site was added to the construct so that the promoter activating the gate can be easily changed. EcoRI restriction site is found at the beginning of the prefix, and by a simple restriction reaction with the two enzymes we could take the promoter out and replace it with a different one.

But why do we want our bacteria to produce AHL molecules? How does it help us to see if a toxin or an allergen is present in our sample?
Continue reading about gate 2 and 3 to find out

Gate 2

Built and Biobricked (BBa_K1343003)

The gate serves as a sensitivity tuner, allowing us to control on the timing of the measurement. When we want to start our measurement we just need to add IPTG to the bacteria solution.

The gate consists of the Plac promoter and the LuxR gene. When IPTG is added to a sample containing our bacteria, LuxR gene is transcribed. LuxR is a transcriptional activator that binds directly to the AHL molecules, so that a LuxR-AHL complex is formed.

And what happens in our system when the complex LuxR-AHL is formed?
Read the next sections about gates 3 to find out

Gate 3

Built alternative construct

Gate 3 enables us to see if our material of interest is present in a random sample by producing GFP and amplifying the signal.

This construct is the final part of our alpha system.
The promoter of the gate is the Plux promoter which undergoes activation when the complex LuxR-AHL binds to it. If the promoter is activated we get the production of a reporter gene (GFP) and the LuxI gene.
As we mentioned in the section on gate 1, LuxI transcription results in AHL molecules.

We experienced many difficulties in building this gate. Therefore we built an alternative Gate 2-Gate 3 construct: Pcat_luxR_Plux_mCherry_luxI

In conclusion, the Alpha System is a 3 gate construct that produces GFP when a substance is detected and signals the other cells around it to produce GFP as well

RNA Splint for protein split and assembly - luxI as inspiration and CMR for proof of concept

Our system is made for the detection of substances in very low concentration. It is based on a signal transduction and amplification between the individual bacterium. The major risk in systems like this one is that a false positive result will activate the whole system, rapidly. In order to solve this problem, we came up with few methods. One of those methods is the split of a key component protein, which activates the system: LuxI. It allows the communication of an activation signal to between all of the cells in the sample. We thought it would be feasible to solve it by ensuring the production of LuxI only when a toxin is in the sample. To avoid the leakiness of the promoters we have decided to split the LuxI gene so each part will be regulated under a different promoter (of the same type). This way the probability for leakiness of the two promoters in the same cell and assembly of the two parts of the protein is very low. That way LuxI will be produced only when the cell detects a toxin.
To screen for the positive colony with the positive RNA splint and the functionality of LuxI, the detector strain assay will be done (the same screening method from gate 1)

LuxI-Inspiration

RNA splint: bridges between A protein RNA and B protein RNA. The splint enables the ligation of two RNA molecules. In order for the ligation of the 2 RNA molecules to occur a T4 RNA ligase is needed. T4 RNA Ligase is the enzyme from the phage that makes this ligation between two RNA molecules, it recognizes a specific site (this will be addressed later on). This is the preferable method.
The design using luxI:

CM Resistance

RNA splint is an in-vitro method described for the ligation of 2 RNA molecules. (M.R. Stark, J.A. Pleiss, (2006)
For a simple and robust screen CM split is preferred over the split of luxI

Some background about the components needed for the described system

• Role of T4 Ligase in nature: to repair tRNA damage during the invasion of the bacteriophage (maybe cause of different anti codon usage in the phage) (“Thus, reprocessing could be yet another T4 device to adapt the translation apparatus to post-infection codon usage”) (Amitsur et al., 1987)(C. Kiong Ho, Li Kai Wang 2004)
• Problem: Ribosome will get stuck because of the double strand RNA
Solution: RNA Helicase-exist in E-coli K-12
• The 5’ of the mRNA is phosphorylated - it is important for the ligation
• The terminator should be removed from the first part of the CM resistance, or else it will be translated in the middle of the gene-will be done by Hammerhead Ribozyme
• Chloramphenicol acetyltransferase is the protein for the resistance - There is a His residue in the C-terminus that is important for the mechanism of the enzyme so it is possible to knock the activity out by splitting the sequence
• We used Rnl1 for this ligation (ligates tRNA)(T4 nucleic acid ligases: Bullard & Bowater 2006)
• It's enough to use 7 bases for each side (14 total) we did 20bp totally (E. Paredes et al. / Methods 54 (2011))
• GTN for the Ribozyme cut, N needs to be purine (but could be any base except G)
• TTC is the loop of tRNA-Lys (UUC) - Needs TTCN for the T4 Ligase
• The first part ends with TTCGTC
• In luxI there is TTCTC so it is supposed to be suitable for the approach
• This is Type 3 Ribozyme.

For extended information follow the link

Bio-Builder: Can we build it? Yes We Can!

Throughout this project, we had many genetic constructs to build, including: the alpha system, the beta system, the RNA splint, and testing Toggle Switches in order to optimize the beta system. As you may know, building these circuits is not easy: it requires a lot of lab-work, and has a high rate of failure, in addition to the fact that each reaction requires a different method, and individual planning.
To solve these problems, we designed a new standard for the iGEM community, which will allow teams to construct many different parts, using only a small set of almost identical Gibson reactions which we have shown to have a 94% success rate over the construction of 21 gates. In addition, because these reactions have been standardized, they are compatible with high-throughput methodologies. Finally, we implemented a simple method for making existing parts fit our standard.

The Standard

Our standard is based upon a set of Gibson Addresses – "barcodes" which we have strategically placed along the genetic sequence, between the different promoters and genes. It is possible to use these barcodes (which have been optimized for PCR and Gibson reactions), to amplify pieces of DNA by PCR, and combine them using Gibson reactions. There are two types of parts in our standard:

How to build a circuit

This allows us to construct any one of the following constructs:
                
                

Where P is any promoter in our system, G1 is any gene of class 1, and G2 is any gene of class 2. To see how we build any one of the constructs click on its image. *Figure*

In addition, it is quite simple to convert genes into our standard:
Just PCR the desired gene with barcoded primers and then Gibson with any other construct
In addition to designing a standardized methodology, we also optimized the sequences of our barcodes for PCRs and for Gibson Reactions. This is evident from the success rate.
Overall, using our new method, we have successfully built 21 genetic gates (of which 12 were bio-bricked), and only 1 reaction failed along the way, giving us a success rate of 94.2%. As a control we did 14 Gibson reactions using the iGEM prefix and suffix, of which 6 were successful (and bio-bricked), giving it a success rate of 42% (meaning that a Gibson reaction with the iGEM prefix and suffix is 10 times more likely to fail than a Gibson reaction using our optimized overlaps).
See the full list of all the constructs built using our methodology, which addresses were used, and which templates were used:

*mCherry contains restriction sites which makes it unbio-brickable

For the sequences of the barcodes, and their implementation in our project, see our bio-bricks page