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Basics Regulatory Systems The Switch Anti-Repressor group Anti-Terminator group

Background What is Synthetic Biology? Not many people have heard of Synthetic Biology. Synthetic biology essentially aims to utilize natures tricks to design and build artificial biological systems for engineering purposes as well as a way to get a better understanding of why biological systems are set up as they are. The term “synthetic biology” was first used on genetically engineered bacteria that were created with recombinant DNA technology. Parts from natural biological systems are taken, characterized and simplified and used as a component of a highly unnatural, engineered, biological system. The term was then used when referring to when organic synthesis is used to generate artificial molecules that mimic natural molecules such as enzymes. There are two types of synthetic biologists: those who deal with re-designing and fabricating existing biological systems. those who deal with designing and fabricating biological components that do not already exist in the real world. Synthetic biology provides us with a new perspective from which we can understand and ultimately utilize life for our own benefits. [1,2,3] Introduction Aim The goal of our project is to enable colonies of ''E. coli'' bacteria to transition between production of two different reporter proteins. In our system, switching between states will be induced by exposing the bacteria to light. Each of the states will have a specific frequency associated with it. There are multiple potential applications for biologicals "switches" such as these, this includes the improved control of production of additives in industrial biotechnological processes. Project Concept As previously stated, the main goal of our project is to design a bistable switch. The switching between the two states will be controlled by the introduction of two different wavelengths of light, each wavelength responsible for the induction of a different state. As a proof of concept, we’re using fluorescent proteins as reporter genes which makes it easy to observe and characterise the system. In principle, however, any reporter gene can be used. Our original project concept revolved around using light-receptors to instigate the switch between the two stable state. It was thought that the production of the first reporter protein would be induced by red light (660 nm). At the same time, production of the other reporter will be suppressed by a coexpressed repressor. Conversely, production of the second reporter would be induced by blue light (470 nm). Bistability of the system is achieved by using two repressors which negatively regulate each other’s expression. This enables the system to sustain state without continuous input, i. e. once production of a reporter protein is initiated, it will persist until the system is forced into the other state. Our project concept has since changed to using two different carbohydrate sources as a means of switching between the two stable states. This means that the state in which the bacteria will be found depends on which one of two carbohydrate sources it was last exposed.

Micro fermentor systems

description of the biolector and a few references

Synthetic promoter library (SPL)

How does it work, examples, what have it been used to characterize? how do you construct it? Figures and illustrations to explain. Figures to explain our use? And example on our specific design primer sequences illustration on the double stranded DNA, with BB - prefix suffix.

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 Unsertainties 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.

Presentation

Figures and illustrations - explanations

Designing the switch - selecting parts

(modeling) 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.

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.

Flourescene protein

Why were the shown proteins selected.

The Biolector

selected filters in releation to flourescence protein
The filters for the biolector is ordered individually to fit the required needs and proteins. we decided to order the filters we needed for this application.

Filters applied for this experimentWe chose RED: Green:

Characterizing BBricks as parts of the switch

(Materials and Methods) section

Mainly the hard control of the switch is due to a double regulation system build on a both terminator-anti-terminator and repressor anti-repressor regulation. It was out of the scope of this project to construct the entire theoretical developed switch, and characterize the fully constructed switch. Have focused on characterizing the two regulatory systems individually. This was done in order to investigate if the responses were satisfactory to use in a future complete switch construction. (???? By getting the regulatory mechanism of the subparts we further, by modeling, could conclude constraints for successful function of the system and other subparts. In this section we describe the design of and the experimental setup used to characterize the subparts of the system and our bio-bricks.

Anti-terminator function

(experimental work)

selecting supparts

Why were these supparts chosen ?
AIM

Design and experimental setup

presentation - Figure of setup and explanation

Materials and methods

HOW ? what plasmids and why, what measurering method and why? refer to the notebook page with protocols - and actual info from lab.

mRNA-stability when introducing non-coding sequences problems will acour with rna-degredation of RNAP is not attracted to the area, to fast degredation, unwanted steam loops. (Reference to the terminator screening plasmids for BB)

Synthetic promoter library (SPL)
How does it work, examples, what have it been used to characterize? how do you construct it? Figures and illustrations to explain. Figures to explain our use? And example on our specific design primer sequences illustration on the double stranded DNA, with BB - prefix suffix.

Results

comments to the results and reference to the BB pages with info and results.

Repressor function

(experimental work)

selecting supparts

Why were these supparts chosen ?
AIM

Design and experimental setup

presentation - Figure of setup and explanation

Materials and methods

HOW ? what plasmids and why, what measurering method and why? refer to the notebook page with protocols - and actual info from lab.

Results

comments to the results and reference to the BB pages with info and results.

References

• (Gottesman et.al. 2002) Gottesman. Max E, Nudler. Evgeny, 2002 ”Transcription termination and anti-termination in E.coli” Genes to cells. (a good introduction review to termination function)

• (Franklin et.al. 1989) NC Franklin, JH Doelling - Am Soc Microbiol "Overexpression of N antitermination proteins of bacteriophages lambda, 21, and P22: loss of N protein specificity." - Journal of bacteriology, 1989

• (Jensen 2004) Ole Nørregaard Jensen, “Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry,” Current Opinion in Chemical Biology 8, no. 1 (February 2004): 33-41.
• [1] http://syntheticbiology.org/FAQ.html
• [2]http://www.nature.com.globalproxy.cvt.dk/nrg/journal/v6/n7/execsumm/nrg1637.html
• [3]http://www.nature.com.globalproxy.cvt.dk/msb/journal/v2/n1/full/msb4100073.html