Team:DTU-Denmark/Switch

What is a switch?

what is a biological switch, examples and existing constructions, What can we use it for. what have been build. Many small cuircuits have been constructed. And reviews have been done on also trying to build regulatory function in enzymes. See article (“Designing switchable enzymes” Marc Ostermeier)

Design and engineering of bi[o]stable

The THEORETICAL overall aim and vision
(THE bigger picture and STORY)
APPLICATIONS
how we designed our switch - selection of parts and parameters - and last presentation of our system.
Applications
Uncertainties and potential problems:
By designing the switch we did not know the exact distance needed from the nut-site to the terminator steam loop for proper function of anti-termination. We have taken the sequence form the natural seting and made it small enough to give sense as a biological building block.
We have tried to utilize the phage regulation to construct a biological switch that can be used in biological engineering.
When considering how to characterize the subparts of our system we looked at the work already done to characterize terminator efficiency. The screening plasmids made by (REFF) endy and XXX and XXX. The work clearly demonstrates the problem by creating a weldefined data sheet system, the data achived in terms of terminator efficiency is not consistent, and shows the complexity of biology.
It has not been possible for us do all the test needed to develop a wel defined switch system. Below is outlined our approach and in the end we suggest other approaches and possibilities for further work, and considerations in relation to this.

Selection of Parts

Requirements before a biological switch functions. On the paper and theoretically.
Components of the switch
We have decided not to use cI, why? The hong kong paper flaws!! (REF) we did not use UV-activation why? To have a stable system we did not what to use cI and UV-regulatory systems as they can impose problems with the genetic stability.

Modeling

(modeling)

Selecting N protein and nut site

In the end, after evaluating what component pair to use we selected λ N-protein and nut-site. Different nut-sites N-protein systems have been identified and investigated (REFFF), the nutsites for λ-phage and p21, p22, are the best described (REFFF) comparison of the antiterminator effect have not been charfully investigated, as emphasis have been on function and interacting parts. we wanted to selected the nutsite with a strong consistent anti-terminator effect. But as this was not well defined continues work was done with the lambda nut side because more articles and knowledge was available, for potential trouble shooting and improvement of the system interaction and dynamic.

What have been described is that the N-nut-site pair have specific function and thus the λ-N-protein was used for continues, construction of the switch.

Fluorescent Proteins

as we departed in the idea from the terminator screening plasmids described in the partsregistry.org (REFFF) we had a primary focus on fluorescence proteins as our reporter systems. Further we wanted to have high quality data, with a high resolution. We descided on the in-house expertice on using a continous microfermentor system that can measure two fluorescence proteins continuously (biolector) and a flow cytometer, also capable of measuring at two different wave length.