Team:AHUT China/Project

1,The limitations of the electronic computer
With the gradual development of the times, the electronic computer has many advantages such as high automation，fast calculation speed, high precision and memory function is facing more and more bottlenecks. Graph theory problem when the point line with the number of points and lines is increased, Electronic computer that brute force all traversal to complete the form contrast and calculation way, so that when the number of possible solutions to rise to a certain number of times, computer self bearing very big operation pressure; When the chip process approximation of silicon atomsbottom line, electronic circuit high density causes feverand instability makes traditional silicon chip with inevitablyin the face of the calculation problem of the similar path planning of the age of big data when not carrying many difficulties;

2, The establishment of a biological system to solve the problem of path planning
Which can be a good way to solve the computer 'brute force'? Learned that many of the advantages of biological systems and understand the problem of the feasibility, anhui university of technology team is committed to developing a biological navigation system to help ordinary people to complete daily life problems faced by the navigation planning, users still only need to input the starting location, and the needed road conditions, ways and so on conditions, our system can adapt to deal with the complex and changeable and the actual path planning tasks.

3, The rise of biological computer
In the face of a computer to solve a problem, DNA calculation of the unique high parallelism, large storage capacity, low energy consumption and many other advantages manifest. Because of these unique advantages, DNA computing becomes more and more impressive is not surprising.

	DNA calculation	electronic computer
parallelism	The number of DNA treatment under routine experiment for 1018	Traversing the data to compare
The amount of storage	The average length of 4 base pairs along the molecules composed of DNA is only 0.3nm, calculated according to each strand of DNA is 1000bp, the length is only 350nm	The electronic computer is itself limited storage space, even if there is still no way hard disk expansion greatly increased
Energy consumption	Per joule energy can complete 109 DNA coupled reaction	2*1Per joule energy can execute 2*1019 operations

4, Using bio-computer to solve the problem of path planning
Path planning is the most commonly used in our daily life is the navigation software, in addition to this, the road planning, urban network, logistics, communications technology can topology for point, line problem such as routing planning problem basically all issues can be resolved with the path planning; Since biological computer may solve the problem of path planning, whether we can be real problem creature, then be solved, For example: we can the city map point line; (build path elements database) to establish a set of biological information database; calculation of the traditional DNA control in the exponential explosion phenomenon, then run the system so as to solve the existing problems in our.
5, The limitations of the existing biological computer
The calculation of the existing DNA still exists many problems: for example, DNA computing experiments are applied to the solution of a problem for a class of problem specific, universal has no compatibility; without exhaustive algorithm screening makes the calculation of the required DNA exponential explosion; in order to solve the problem of the use of complete biological method in contrast, at the shortest path computer serial computation without effective complementary, but reduces the computation efficiency; the actual navigation planning tasks often have such as the way of location, road type selection and more complex issues to consider, which is currently the basic graph theory research of biological computational models do not have; DNA calculation of these imperfections is precisely the calculation of DNA broad space to explore, and also is the foundation of our research.

The target of our project

Our goal was to establish a new biological navigation system for ordinary people, including the establishment of navigation database, the construct of all path according to the specific situation, the design of finding the right path through the visual fluorescence and so on .Our team designed an operating system based on traffic information (from the hotel we live to the center)in order to explain the specific operation and the feasibility of our project.

The establishment of bio-navigational database

The whole city map was abstracted into a element of the collection constituted by a set of point elements and path elements. The abstracted path elements included all roads in the map and the abstracted point elements included intersections of all roads and some landmark buildings etc. So we designed a set of biological information database. We put the 40bp random single-stranded DNA as the biochemical reaction module of point elements. The biochemical reaction module of <A,B> path element was represented by complementary chain of the second half of the single-stranded DNA represented by point A module and 40bp single-stranded DNA connected with complementary chain of the first half of the single-stranded DNA represented by point B module.
(Database diagram,the city library can also used as node/point library.)

For leaving the interface so as to insert the logical operation module with the level of representation in the subsequent steps ,we designed specific enzyme cutting site which was different from the standard of biobricks.They were used for screening out a suitable path in our project .

The search for suitable paths through the Adelman principle and sequencing

We would refer to the classical Adelman DNA calculation in Hamilton path so as to help us find all possible pathways.
At first, we selected all the coding modules that computation module needed . These coding modules would form DNA coding collections of modules with combination of any cities and any paths by ligase.Then ,we used the first half of the start node's complementary strand and the last half of the terminal node's sequence as the primers. And then do a PCR reaction for these pathway sequence,only leaving the pathway of starting point and end point.

After doing electrophoresis ，we gained possible DNA sequence in suitable length, then getting DNA sequence the of the first step through a plastic recycling. Finally , we obtained the specific results of pathway by sequencing

The detection of the pathway that our project needed In visual view

After thinking of the traditional screening methods in front, we further attempted to screen out the pathway containing the sequences of starting point, end point and other points on the way by the level of gene expression. According to the relevant data, we designed a logic gate expression system for the path planning problem in order to help us to detecting the pathway our project needed through the visual angle. We chose GFP DEVICE as a carrier to realize visualization after comprehensive analysis. The Above-mentioned design narrowed the scope of screening, so we used it in specific enzyme cutting site different from the standard of biobricks, then sequencing and analyzing the results of screening.
(The path way sequence will insert in the plasmid by designing the Biobrick.'s prefix and suffix. And then we can use it to do the following test)

The theory

According to the references, we build an constructed an in vivo synthetic-hybrid system（BBa_K1341000）.The promoter of the GFP has −35 and −10 sequence. A lac operator site is upstream of the −35 sequence, a tet operator site is between −35 and −10 sequence, the other tet operator site is downstream the -10 sequence. This promoter can be restrain by the LACI or TETR proteins. We can engineer the system in an Escherichia Coli strain that constitutively expresses lactose repressor (LacI) or tetracycline repressor (TetR) proteins at the same gene circuit. The output fluorescence is then dependent on the input of two small molecule inducers, anhydrotetracycline (aTc) and isopropyl-_-thiogalactoside (IPTG). IPTG and aTc interact with LacI andTetR, respectively, which then free the operator sites for RNAp to bind and initiate transcription. (there are many kinds of engineered GFP promoters can be design in this device, but the LTT promoter have the more apparent GFP expression than other kind of promoters according to model figure in result page.)
This principle can help us build a logic device for our bio-compass.

Operating steps(judging whether a pathway contains three specified nodes at the same time)

1.  Trying to insert the LTT GFP device(BBa_K1341000) in the test circuit(the plasmid involved possible path way sequence) by using the AFL III enzyme site. If this circuit involve the N0.5 node, we can detect the green fluorescence.(Each step will reject the test circuit which result out of expetation)And we make the 2-4-5-7-8 path way sequence which has insert the LTT GFP device as an exemplary test system(BBa_K1341001) to do the following test.

2.  Trying to insert the tetR device (BBa_K1341003) in the test circuit by using the BamH I enzyme site. If this circuit involve the No.2 node, it's GFP device will be restrained by the tetR protein.

3.  Trying to insert the LACI device(BBa_K1341002) in the test circuit by using the Nco I enzyme site. If this circuit involve the No.8 node, it's GFP device will be restrained by the lac I protein.

4.  Inputting the small molecule inducers IPTG first, if the test circuit has both tetR device and LACI device, it will still be restrained by the tetR protein.

5.  Then Inputting the small molecule inducers aTc, if the test circuit has N0.2, N0.5, N0.8 node. Dependent on the input of both two small molecule inducers, the output fluorescence will change as expected.

(The model and the characterization about this test will see in the result page.)

According to this test, we can define whether this path way sequence is match users' condition (such as start from No.2, pass No.5, end at N0.8) first. Then we can confirm the specific route of this path way sequence by using DNA sequencing technology.

Summary:  Biology Computation is a new field, we attempted to construct a biological navigation device which contains DNA calculation and the function of gene logic operation. Combining with the engineered thought of synthetic biology and integrating various biological calculation, we tried to dig out more practical Value of computational biology.

Electrophoresis

This is the result of the possible pathways' connection. We can choose the possible pathway's DNA sequence to make the gene circuit and insert it in the plasmid to do the following test.

We can prove that the GFP signal part(BBa_K1341000) of the screen device has been insert in our path way test system((BBa_K1341000 is almost 1000bp, and the path way sequence is about 200 bp, this Electrophoresis result is about 1200bp), and we make this gene circuit(BBa_K1341001) do the follwing test about whether this path way circuit is the right way we want.

Model

This model tells that the BBa_K1341000 with elements of the tet, lac, they will compose as a device and is responsive to the commonly used inducers IPTG and aTc, producing GFP as an output signal. So it can prove that the choice device can help us build a switch system for bio-compass. It can help us screen out the possible path way which include the start point, terminal point, and the particular intermediate nodes (such as the (No.5 node) in our story).

The x and y axes form a grid of 36 pairs of inducer concentrations. The concentrations tested were 0, 1, 10, 50, 100, and 200 ng/mL for aTC and 0, 0.001, 0.002, 0.005,0.010, 0.100, and 1.000mM for IPTG behavior is depicted 6 h after induction.

Characteristic

In this graph, we can get the conclusion that the fluorescence level still has an apparent change when we add aTc firstly. So, we decided to add IPTG firstly and then add aTc secondly. Thus, we can get obvious fluorescence units. Finally, we can judge this path way gene circuit is exactly we need by this way.

There is a characteristic about the LTT promoter in this device, the figure can explain why we first use IPTG for testing first in our project.
Conditions are ±100 ng/mL aTc and ±1mM IPTG, This panel depicts an LTT promoter design, where the single lac operator site is upstream of the −35 sequence. In this case, LacI is no longer able to entirely suppress transcription and an intermediate level of induction (leakiness) occurs at conditions of high aTc but no IPTG. All data is obtained 6 h after induction.

Conclusion

According to the above logic tests and the finally DNA sequencing, we can confirm that the route: 2-4-5-7-8 is a suitable result for us to go to the convention center from the hotel if we want to visit the museum on our way.

Future work

Build a Hierarchical Communication system between BIO-COMPUTER & Electronic COMPUTER
Combining the advantage of DNA or Biology's parallel computing and the modern Electronic COMPUTER technology, Sensor Technology, cloud computing, we can Build a Hierarchical Communication system between our BIO-COMPASS and Electronic COMPUTER. This will make the BIO-COMPASS become a real brand new bio-navigational system that can be used by the ordinary people in the future.

Our submission parts:

BBa_K1341000

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We use this device as a part of a switch device designed for our bio-compass.It can help us screen out the possible path way which include the start point,terminal point, and the particular intermediate nodes(such as the museum(No.5 node)in our story).It has a LTT Promoter,with elements of the tet, lac, they will compose as a device and is responsive to the commonly used inducers IPTG and aTc, producing GFP as an output signal.If this device and tetR(BBa_K1341003)&lacI(BBa_K1341002)successful inserted in the path way by using the typically enzyme site. It will drive expression of green fluorescent protein (GFP). This characteristic will help us to screen out the possible way we want to find.PS: We build a measurement system to test the screen functional about this device in our project(BBa_K1341001).

BBa_K1341001

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This is a measurement system comprised of a switch device(output GFP as signal)and a specialized path way sequence.This system can prove that we have insert the BBa_K1341000 in the testing path way successfully(we designed a AFL III Restriction Enzyme cutting site in the No.5 node sequence. Before we insert the device, we linked the matching AFL III Restriction Enzyme cutting site sequence on switch device's(BBa_K1341000)ends by using BioBrick assembly method.).Besides, this system also has a BamH I Restriction Enzyme cutting site in the No.2(start) node's sequence, a Nco I Restriction Enzyme cutting site in the No.8(terminal） node's sequence. With the same method, the TetR device (BBa_K1341003) and LacI device (BBa_K1341002) can insert in this measurement system according to the requirement. By using the aTc and IPTG, we can test whether the path way is the right result exactly such as this(2-4-5-7-8).

Improve part BBa_K091101
We change this part as an LTT pTet_ Lac hybrid promoter in our device BBa_K1341000,it will have the more active GFP expression than this kind of TTL promoters(BBa_K091101) according to model figure in result page. So we can test the change of the GFP expression more easily.

More information please see our parts form:

		Name	Type	Description	Length
	W	BBa_K1341000	Composite	switch device(LTT gfp)for our bio-compass	945
	W	BBa_K1341001	Measurement	PATH WAY TEST SYS IN BIO-COMPASS	1171
		BBa_K1341002	Composite	LACI in logic gate	1351
		BBa_K1341003	Composite	TetR in logic gate	883
		BBa_K1341004	DNA	BamHⅠ site	10
		BBa_K1341005	DNA	Afl Ⅲ site	10
		BBa_K1341006	DNA	Nco Ⅰ site	14
		BBa_K1341007	Composite	Afl Ⅲ site<TGFP&AFL III SITE	981
		BBa_K1341008	Plasmid_Backbone	pUC57 Plasmid_Backbone	2710
		BBa_K1341009	Composite	GFP that can switch logic gate promoter	927
		BBa_K1341010	Composite	OUTPUT SYS(GFP)LOGIC GATE IN Graph Theory 2	1004
		BBa_K1341011	Composite	OR LOGIC GATE IN Graph Theory (GFP OUTPUT DEVICE)	937
		BBa_K1341020	DNA	solution24578	200
	W	BBa_K1341022	Regulatory	LTT promoters+rbs	82
		BBa_K1341023	RBS	LTT-rbs	13
		BBa_K1341999	Temporary	SYS WITH GFP not