Team:BGIC China/Description
Genetic Boolean Logic Gates
Our BioChip involves the de novo application of DNA recombination which enables the genetic modules to mimic the functions of boolean logic gates in eletricity.
Recombinase like Bxb1 and PhiC31 are able to invert or excise DNA based on the orientation of recognition sites which flank the target gene [1]. With the aids of inducible promoters such pCpxP and pFadR it is possible to express target genes by using boolean logic. It is demonstrated by Piro's team that recombinase based genetic logic gates are capable to mimic all the 16 types of logic gates (Fig.1).
Fig.1 16 different types of synthetic genetic boolean logic gates
Picture from Siuti, Piro, John Yazbek, and Timothy K. Lu. "Synthetic circuits integrating logic and memory in living cells." Nature biotechnology 31.5 (2013): 448-452.
In order to further explain and demonstrate the application of genetic logic gates, we design a simple genetic circuit involves three plasmids which can achieve following algorithm (Fig.2):
Fig.2 A three states logic algorithm; 3 different colored fluorescence proteins will be expressed based on 3 different situations
Fig.3 Genetic circuit which achieve a three states algorithm
When there is only molecule A present, Bxb1 recombinase will be expressed (Fig.3). Bxb1 attached to it's recognition sites and inverts the genes. RFP gene is flanked by two recognition sites of Bxb1 (Fig.3) so the gene will be inverted and be expressed.
When there is only molecule B present, phiC31 recombinase will be expressed (Fig.3) which resulting the expression of CFP.
When molecules A and B are both present, phiC31 and Bxb1 will be expressed. Promoter and CFP gene on Plasmid 1 (Fig.3) will both be inverted so there will be no expression of CFP. Same situation happened with promoter and RFP on Plasmid 2 (Fig.3) so RFP will not be expressed as well.
However, when both promoter and GFP on Plasmid 3 (Fig.3) are inverted, the expression of GFP will start.
Paper based cell-free BioChip
Fig.4 Schematic procedure to make paper based cell-free BioChip
Picture modified from internet
Fig.4 demonstrated a schematic procedure of how the paper based cell-free BioChip is made. We genetically engineered the plasmids by using standard cloning methods (MCS, MoClo, Gibson, Golden Gate, etc.). The plasmids formed a genetic circuit which contains an sensor unit (inducible promoters), a logical processing unit (genetic logic gates) and an output unit (reporter genes like genes coding for GFP, LacZ, Renilla luciferase, etc. Fig.5), which together act as a micro-computer.
We then mixed these plamids with S30 Cell-Free expression system which can be bought from Promega, and immobilized the plasmids with cell-free system onto nuclease-free filter paper.
Freeze-drying technology is used to dry the filter paper[2] while keeping structures of polypeptides and polynucleotides intact.
Freeze-dried BioChip can be then be stored in dry condition for a long time.
Fig.5 Genetic micro-computer
The use of the BioChip
In order to make the BioChip more user-friendly, we design a three-component reaction unit (Fig.6) which contains a loading area for clinical and laboritory substances, a microfluidics conductor that controls the quantity of target molecules and filters unwanted substances that might influence the result, a reaction center which contains the freeze-dried immobilized genetic circuits and substances in S30 system.
Black area surrounds the reaction unit is composed of wax which restricts the flow of liquid.
The BioChip will be placed in vacuum box at low temperature for storage.
Fig.6 Three-component reaction unit
When you need to use the BioChip, you just need to take it out from the vacuum box and load the liquid substances onto the loading area. The liquid will flow through the microfluidics conductor and finally get to the reaction center where all the genetic and logical processing reactions take place.
Fig.7 Signal output for detection of different molecules
Fig.7 is a demonstration of the use of the BioChip with specifically design genetic circuit in the reaction center. When clinical substances contains only glucose is loaded, CFP protein will be expressed. When only fatty acids present, GFP protein will be produced. When glucose and fatty acids are both present in the clinical substances, neither CFP nor GFP protein will be expressed but there will be RFP protein produced.
Therefore, by looking at the color of the fluorescence protein produced in the reaction center, we can easily deduced the biological molecules present in the clinical substances.
In order to increase the throughput of reaction, the BioChips are made into assays which contain serials of reaction units (Fig.8).
Fig.8 Synthetic picture of BioChip assays
Fig.10 3D model of new design of the BioChip
Fig.11 3D model of new design of the BioChip
In the future, we will made our BioChip thicker to contain higher volume of liquid for persistent reactions. Glass cover will be included to lower the evaporation of liquid and contamination of nuclease in the atmosphere (Fig.10 & Fig.11).
