Overturn the traditions
The first programmable BioChip ever
You might have experience of coding on a computer to make a new program, but have you ever thought about to accutually pragram a BioChip to let it fulfill any biochemical tasks you can imagined?
Since the NGBC is completely constructed by genetic circuits, you can just use subcloning techniques to edit the genetic circuits and genetic boolean logic gates to construct logical networks and algorithms.
This is one of the most important feature of our BioChip. S30 Cell-free system eliminates biochemical reactions inside the cell that may influence the result and makes the expression of special proteins, which are toxic to the living-cells, becomes possible.
More importantly, it tremendously increased the bio-safety of our BioChip since there is no worries about the mutations of living-cells and the escaping of harmful organisms.
We will develop an NGBC kit that could be distributed to the labs around the world, so everyone could re-program our biochips and produce their own one. By sending their genetic circuits to a special gene bank and uploading results onto NGBC database, thousands or even millions of biochips with different functionalities that resolve problems in different fields could be created and stored.
Short prototyping time
By using our NGBC kit, it is possible to construct a new biochip just within a week!
One-step and high-throughput
Multiple obstacles in clinical testing procedures could be resolved by using our biochips. Forget about complex and time-consuming sample testing experimental procedures, what you need to do is just adding samples onto a properly programmed NGBC and all the biochemical reactions could be done automatically. Densely packed reactions units on the biochip ensures that large amount of samples could be tested together.
The high-throughput of the bio-chip and the small duration of the testing process of clinical samples, should the bio-chip be successfully manufactured, shall relieve millions of patients of comprehensive time-consuming and perhaps agonising medical inspections—these could be carried out at home! The bio-chip system, presumably, transcends mainstream medical inspections of clinical samples in means of efficiency and convenience.
We introduced an Utra-High Expression Cassette(U-HEC) into our standard NGBC kit which enables controllable high protein expressions.
Our BioChip enables users to fulfill any of their fantastic and creative idea by using standard subcloning techniques.
Read more about our future projects with NGBC by clicking here
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 . 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 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).
Open sourced NGBC kit
We are going make a kit called NGBC kit which contains all the things for you to make a new NGBC. The kit is completely open sourced, you can know the design and the nucleotide sequences of all the plamids and genetic circuits inside it.
We will distribute our NGBC kit to the laboritories all around the world. NGBC database will be made to record all the new genetic logic processing units, genetic sequences and the design of all the new units. We also strongly suggest the iGEM committe to take this as a new trial in iGEM.
Here we made a list of things in our beta version of NGBC kit:
- AHL inducible promoter flanked by MCS in pSB1C3 backbone
- aTc inducible promoter flanked by MCS in pSB1C3 backbone
- Bxb1 recombinase coding sequence flanked by MCS in pSB1C3 backbone
- phiC31 recombinase coding sequence flanked by MCS in pSB1C3 backbone
- Cre recombinase coding sequence flanked by MCS in pSB1C3 backbone
- Bxb1 recombinase recognition sequence flanked by MCS in pSB1C3 backbone
- phiC31 recombinase recognition sequence flanked by MCS in pSB1C3 backbone
- LoxP sequence flanked by MCS in pSB1C3 backbone
- 16 different types of genetic logic gates with EGFP as output in pSB1C3 backbone
- pCpxP promoter flanked by MCS in pSB1C3 backbone
- EGFP coding sequence flanked by MCS in pSB1C3 backbone
- RFP coding sequence flanked by MCS in pSB1C3 backbone
- CFP coding sequence flanked by MCS in pSB1C3 backbone
- Renilla luciferase coding sequence flanked by MCS in pSB1C3 backbone
- LacZ coding sequence flanked by MCS in pSB1C3 backbone
- X-gal in powder form
- J23119 promoter flanked by MCS in pSB1C3 backbone
- B0015 terminator flanked by MCS in pSB1C3 backbone
- pSB1C3 linear plasmid backbone
1. Siuti, Piro, John Yazbek, and Timothy K. Lu. "Synthetic circuits integrating logic and memory in living cells." Nature biotechnology 31.5 (2013): 448-452.
2. Pardee, Keith, et al. "Paper-based synthetic gene networks." Cell 159.4 (2014): 940-954.