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.

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sRNA

(undetermined)

37°C RBS

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

The system we have created in this project is consisted of two different parts: light induced part and dark induced part. Each part regulates two different genes, resulting in a total of four genes that can be regulated. By adding new parts to the system, it is plausible that the system can regulate more genes. We tend to do that by employ more light sensing promoter. Each light sensor we add, we would be able to regulate two more different genes by employing regulation mechanism of 37oC RBS and sRNA.

On the other hand, we tend to optimize our system to achieve subtle control of the four gene expressions. We might be able to do this by taking the values between the limits we have set. Instead of 30oC and 37oC, we tend to take the values between so we can create a lot more conditions under which different level of expression can be achieved. We can also vary the intensity of red light to control the level of expression. The ultimate goal is to precisely control the level of expression of each gene.