Team:NCTU Formosa/project

Introduction

The main aim of our “E.coline” project is to generate isobutanol, a promising eco-fuel, in a productive and efficient way.To produce isobutanol, at first we use four pyruvate catalytic enzyme genes: alsS, ilvC, ilvD, kivD all. We then designed a temperature control system to allow E.coli to produce optimum isobutanol before being poisoned by isobutyaldehyde. According to our data(Figure 8), our temperature control system had been proven to work successfully. Furthermore, in order to produce isobutanol more efficiently, we combined zinc fingers and our enzymes together and put the fusion proteins in catalytic pathway order, thus the isobutanol conversion process can be accelerated. Besides, our cellulose test result has proven that in our project we are truly able to convert agricultural trash into the precious isobutanol.

[http://30cm.me/igemc See the page]

sRNA

(undetermined)

37°C RBS

http://fitisafeministissue.files.wordpress.com/2013/03/orange-juice.jpg

A

The main aim of our “E.coline” project is to generate isobutanol, a promising eco-fuel, in a productive and efficient way.To produce isobutanol, at first we use four pyruvate catalytic enzyme genes: alsS, ilvC, ilvD, kivD all. We then designed a temperature control system to allow E.coli to produce optimum isobutanol before being poisoned by isobutyaldehyde. According to our data(Figure 8), our temperature control system had been proven to work successfully. Furthermore, in order to produce isobutanol more efficiently, we combined zinc fingers and our enzymes together and put the fusion proteins in catalytic pathway order, thus the isobutanol conversion process can be accelerated. Besides, our cellulose test result has proven that in our project we are truly able to convert agricultural trash into the precious isobutanol.

B

Overall, we’ve completed our whole project of utilizing glucose to produce isobutanol and reached a production rate which is higher than recently published papers (0.8%=6.4g/L). Furthermore, we have also realized our dream of “Changing Trash into Gold” which is converting agricultural trash into isobutanol.

Light regulator

Pathway Regulation

Our system is consisted of two independent parts: light induced part and dark induced part. Each part is capable of expression two different genes depending on the conditions in which the bacteria are grown; and therefore, a total of four different gene expression can be regulated.

Light induced part

At 30°C , Pred is activated by red light and translation proceeds. However, ribosomes can only bind to the normal RBS, as 37°C RBS forms a hairpin structure, forestalling ribosomes from binding. The only gene downstream of the normal RBS is RFP, so only RFP would be expressed. On the other hand, 37°C RBS unfolds at 37°C , resulting in the expression of both LuxR and GFP. LuxR would binds with AHL to form a complex that activates Plux. The activation of Plux produces sRNA that binds to the normal RBS, blocking it from ribosomes. As a result, RFP would not be expressed and only GFP remains at 37°C .

Without red light, Pred would not be activated, and therefore, this system is completely shut down in the dark.

Dark induced part:

Pred activates in the presence of red light, producing tetR that represses Ptet. In other words, the dark induced part is inactive in the exposure of red light and active without red light. At 30°C in the dark, only normal RBS functions to express YFP while 37°C RBS remains as a hairpin structure. At 37°C, however, 37°C RBS unfolds to express LuxR and BFP. Just like the mechanism employed in the light induced part, LuxR forms a complex with AHL to activate Plux that produces the sRNA to block normal RBS. This way, the YFP downstream of YFP cannot be expressed and BFP is expressed at 37°C

Future Work

(undetermined)