Difference between revisions of "Team:HUST-China/Part Collection"

Collection List

Here is the list of our part collection:

The part collection well documents the prokaryote regulation circuit of our project with every detailed characterization of the transcriptionally related proteins and promoters.

The whole circuit employs bacteriaphage lambda operon and is redesigned as a positive-feedback tri-stable switch. Users can define two inputs and three kinds of outputs to make a sense-response system or an analysis-report system.

To characterize the internal protein-protein and protein-promoter reactions, we build four sets of test circuits:

Cro and pRM

1.Cro and pRM inhibition test:

The control group and test group are seperately shown as below:

We inserted the circuit into PET-Duet-1 palsmid and used plate reader to see the flourescence difference.

We can see from the figure above that the flourescence of test group went down and seperated clearly from the control.

CI and pR

2.CI and pR inhibition test

We used the same method as Cro and pRE characterization to test the inhibition of CI and pR. From the figure above we can clearly see that CI does reduce the expression of GFP.

And we also did fluorescence microscope detection after 30, 120 and 240 minutes induction.

From pictures above,the fluorescence of both two groups was increasing over time and it is obvious that the test group which contains CI expressed less GFP protein than control group. The results verify the inhibition of CI from pR in a more intuitive way.

CII and pRE

3. CII and pRE activation test

The method of verifying CI and pR inhibition is also applied to test activiation of CII to pRE.From line chart and fluorescence detection,we can see that the test group containing CII expressed more GFP protein than control group.

CIII and Ftsh

4. CIII and Ftsh

Moreover,other characterization circuit in the collection can also serve as an individual part to construct positive and negative control of interest genes.

Tri-stable Switch

The final circuit can serve as a signal filter, and users just need to add two sensors and two interest genes into the circuit. We highly recommend to use our submission plasmid as template and employ In-Fusion methods to achieve the assembly.

To test the switch fetures, we build a characterization circuit with GFP and RFP as reporter genes, ptrp and pT7 as sensors:

In ideal conditions, RFP is supposed to be visualized through fluorescence microscope when induced by IPTG. And after adding IAA into the medium, our host strain will turn to the other state of GFP expression. And because of LVAssrA tag’s effect, the RFP to GFP expression transition will be relatively obvious.

Application Circuit

To provide an application example, we constructed an application circuit based on this version to help relieve lactose intolerance (more details see to application page) .

In the bi-stable switch of prokaryotes, we set the gene of interest 1 as iLDH and gene of interest 2 as beta-galactosidase. For sensing the signal, we choose lactose-inducible promoter plac(mutant) and base-inducible promoter patp2 (works when the pH of microenvironment is about 7~9).

And we hope more teams to join and add more possibilities to the circuit!