Team:NUDT CHINA/Notebook

To solve the first problem, we built devices that contains promoter, RBS, target gene (CDS) and terminator to simulate the nodes and lines in directed graph: the promoters are nodes and the target fragment are lines. When the promoter is activated, the target gene can coding protein which then activate the next promoter as regulatory element. This regulatory process can describe how a man the walking in a directed graph: he departed from the node P1, went through the line L and finally arrived at the spot P2. In this way, theoretically, we can describe any given directed graph in E. coli. (For further details about the coding of the graph, please refer to the project profile of our wiki) (Fig. 1)

Fig. 1 One promoter and one target gene can simulate one node and one line

After building one complete graph in E. coli, different route should be labelled and tested independently so that we can get the length indirectly by the time of whole travel from beginning to ending. Then we can selected the quickest one as the shortest route. However, we are stuck in the second problem: how to build different route.

First design:

In each regulatory unit, three different restrict enzyme sites are put between the promoter and RBS, target gene and terminator, terminator and next promoter, respectively. (Fig. 2)

Fig. 2 the position of three different restrict enzyme sites

In this way, we can cut the RBS and target gene out which means this route does not have line L (we link the 1, 2 (or 1, 3) to keep the circle structure of plasmid). Then we can test this route by the time of arriving at the ending node which reported by the report gene (we used the GFP).

Second stage:

But we found that it was not easy get so many different good restrict enzyme. So we change the design slightly. (Fig. 3)

Fig. 3 the position of two same restrict enzyme sites

In this way, we can still finish the function of first stage but save my restrict enzymes.

Third stage:

The last problem is how to compare the length of different pathway. Firstly, we decide to build all the possible route and test them. That is to say, we need to cut out 1, 2, 3,…,device(s) so that we can test the entire route, which is named exhaustion, quite time-consuming. Later, we found a better way where we can only cut one device which means we only need to build n graph if the graph has n lines.

Provided that we have 5 lines in a graph like Fig. 4.

Fig. 4 a graph of 5 lines, where node 1 is the beginning node 4 is the ending

According our method to find the shortest path, the first step is to build 6 different systems. Then we tested the “finished time” of each systems. Here is the systems and their “finish time”.

System	Array	Time (Theoretically)
11111		2 units
01111		2 units
10111		2 units
11011		2 units
11101		3 units
11110		unreachable

Noticed that without line L5, the ending is inaccessible which implicates the L5 is indispensable. Though it does not block the accessibility, the absence of L4 slows down the arrival, which implies that L4 is on the shortest path. Thus, from the date of the chart above, we can pick out the shortest path is:

Protocol

Here are the recapitulative protocols of the experiments that we have used.

Polymerase Chain Reaction (PCR)

Most of our PCRs was performed with the Recombinant Taq DNA Polymerase TaKaRa Taq(TM) or PrimeSTAR(R) HS DNA Polymerase and dNTP mixture (2.5mM each) under the standard 3-step PCR protocol. Touch-down RCR procedures were also used for several times to inhibit the nonspecific amplification. (Refer to the instruction for more details).

Plasmid Preparation

We used the TIANprep Mini Plasmid Kit (TIANGEN) to perform all of our plasmid preparation on bacteria with target plasmid. (Refer to the TIANGEN instruction for more details).

Gel Electrophoresis & Gel Extraction

After the gel electrophoresis (normal experiment), we used the TIANgel Midi Plasmid Kit (TIANGEN) to extract all our target fragment in gel. (Refer to the TIANGEN instruction for more details).

Endonuclease Digestion & Ligases

The enzymes we had used were EcoR I, Spe I, Xba I, Pst I, Cla I, Sal I, Kpn I (digestion), T4 DNA ligase (ligase) all of them were bought from TaKaRaTM. The digestion and ligase systems were built following the instructions of TaKaRa(TM).

Fluorescent Fusion Protein Gene Expression

Fluorometric measurement were processed with Fluoroskan(R) Ascent FL from ThermoTM using the filters of 485nm and 538nm. The E.coli to be tested was cultured in liquid LB media containing 35μg/mL chloramphenicol to OD0.6, than split into 96 well plates (from ThermoTM) by 150μL/well. The plate was then put into the Fluoroskan(R) Ascent FL and cultured in 25℃ and background shake of 90rpms. The Fluorometric measurement was processed with the interval time of 10 minutes.

Blue print

According our design of the whole cascade regulatory pathway as Fig. 1 showed, we can solve the Shortest Path Problem. However, building and standardizing these devices and systems means drawing the blue print.

Systems	Functional fragments at plasmid
System “111”
System “011”
System “101”
System “110”

Fig. 1 regulatory pathways

Thus, we drew a picture of our experiment procedure. (Fig. 2)

Fig. 2 experiment procedure

Week Events

Following our big plan, we started our daily experiment.