Instructors: Fernando Govantes Romero, Luis Merino Cabañas, Manuel Béjar Domínguez, Antonio Jesús Pérez Pulido, Antonio Prado Moreno, Víctor Álvarez Tallada, Rafael Rodríguez Daga. Students: Adrián Arellano Davín, David Caballero Pradas, Paola Gallardo Palomo, Yolanda Elizabeth González Flores, José Gutiérrez Tabuenca, Jesús Jiménez Sobrado, Amalia Martínez Segura, Aída Moreno Moral, Luis Eduardo Pavón, Félix Reyes Martín.

To store information into bacteria as bits, the first thing you need is a good bistable system. Provided that none of the previously reported flip flop is robust enough, in our project we try to characterize and improve them. So, starting from the characterization (experimental and mathematical and multi-agent modelling) of a basic flip flop, we developed both improved and epigenetic ones. In our Foundational Advances section, we have developed the Mini Tn7 Biobrick tool kit (it allows the integration of genetic information into genomes) which has also been used by our lab in our bistable systems and. Finally, we have also developed the BioBrick Creator (a software tool to facilitate and make accessible for everyone the process of creating and combining biobricks). Several bioinformatic analyses have also been performed to assist and complement our labs’ work. As for Human Practices we have launched the first Synthetic Biology blog in Spanish (Tornillos y Genes (http://www.tornillosygenes.es)), talks in high schools, Science Fairs and more.

Basic Flip Flop

Set of fully biobrick-compatible plasmids for BioBrick chromosome

integration in a wide range of organisms based on Tn7 transposition function. In addition of improving the

regulation of genetic devices by single copy conformation, the miniTn7 tool

generates safer organisms, as horizontal transfer is minimized and the lack of drug resistance markers. The kit also includes a

verified portable Tn7 attachment site that may allow the use of this kit in organisms

lacking the transposon insertion site .

MiniTn7 Biobrick Toolkit

Figure 11. miniTn7, how it works. Steps to integrate a BioBrick into a bacterial chromosome using any of our 10 new plasmids. Find all this tools in the new Genome Integration page in the Part’s Registry.

Table 1. Data of miniTn7 characterization.

Green State

GFP cI (ts) Plac Prm LacI RFP

GFP cI (ts) Plac Prm LacI RFP

Heat sh

ock

IPTG

pu

lse

Red State

E. Coli chassis

Figure 2. Basic Bistable System. This device can be in two different states: when c1ts repressor concentration is high, bacteria expresses GFP becoming green; On the other hand, when LacI concentration becomes high does not allow expression of GFP and c1ts, and RFP is expressed. .

Figure 5. Improved Bistable System. This device has two new modifications: A protease in one side that degrades the repressor and the reporter of the other side, which in turn has an as-RNA that blocks the expression of that protease and the corresponding reporter. With these improvements we want to achieve a more stable system.

Figure 3. Bacteria expressing the bistable. On the left, bacteria are in “red state” while the right

bacteria are in “green state”.

Figure 8. Epigenetic bistable. This toggle switch allows the expression of a reporter gene by decompacting the DNA structure where it is integrated. Through the heterologous expression of a targeted silencing protein, tetR·CSD, the DNA can acquire an heterochromatic conformation, blocking the expression of GFP.

In order to improve robustness and minimize the leakiness of

the basic flip flop, we implemented a dual

proteolysis-asRNA (small antisense RNA) system.

Figure 4. Simulation of the basic bistable system using a multi-agent system based approach. The plot shows that at 30 degrees, one of them is much more stable than the other.

iGEM is much more than just a scientific project!! Talks in high schools, participation in Sevilla’s Science Fair and week of Science. Creation of the first Synthetic Biology blog completely in Spanish. In Tornillos y genes (literally, screws and genes) everyone is welcome to collaborate and can keep up-to-date with the latest

Synthetic Biology news.

Figure 1. Tornillos y genes (screws and genes). It is the first blog about Synthetic Biology in Spanish.

Our project starts by the complete characterization of a basic flip flop by using

several approaches: experimental and modelling (mathematical and multi-agent).

Improved

Flip Flop

Epigenetic

Flip Flop

Flip flop developed in the S.pombe in which the ON/OFF states are controlled

by chromatin remodelation.

Figure 10. Screening of positives clones of Tadh1-tetO2 into pfa6a-MX6-Purg plasmid.

Figure 9. Indirect docking modelling approach to predict the functional state of fusion proteins generated in our lab. Docking models for the TetR-BAMSir3 interaction. The best three shape docking results are displayed on top row while the best shape+electrostatics docking results are shown in the row below. Hex (protein docking server).

ABSTRACT Human Practices

Foundational Advances Biobrick Creator

Figure 12. Multisequence alignment and logo obtained for the attTn7 recognition site, that demonstrates its high level of conservation.

Making accesible to everyone the process of creating Biobricks!

Hosts Delivery methods

Transform. efficiency (transform /ug DNA)

Transpos. efficiency (transpos. /ug DNA)

Transpos frequency (transpos./vible cfu)

Site-specific insertions (checked by PCR)

P. putida

Mating NA NA 1 x 10-4 12/12

Electroporation 6 x 109 7 x 101 NA 10/12

E. coli Heat-shock transformation

1 x 108 4 x 102 NA 11/12

0

50

100

150

200

250

300

0 2000 4000 6000 8000 10000 12000

Pro

tein

(u

nit

s)

Time (seconds)

Basic bistable evolution at 30º

GFP

RFP

Figure 6. Relative fluorescence of the basic and the improved flip-flop during a 42ºC continuous induction. It can be seen that the state switch in the improved flip-flop occurs earlier than in the basic flip-flop. Furthermore, the graphics show a bigger green-to-red fluorescence difference in the improved flip-flop, compared to the basic flip-flop.

Figure 7. Relative fluorescence of basic

and improved flip-flop after 42ºC induction during 11 hours and

without any induction during 6,5 hours. We show that 42ºC state stability is markedly

higher in the improved Flip-Flop compared to the Basic one, and that

the difference between the

fluorescence levels of both states is also

much more pronounced.