E.colightunerMultiple regulation by light, temperature and sRNA

Advisors: Prof. HSIEN-DA HUANG, Assist. Prof. WEN-LIANG CHEN, Assist. Prof. HSIAO-CHING LEEInstructors: CHIEH-CHENG YU, CHENG-HSUAN WU, TING CHANG, MALVIN JEFRI, SHU-HAN CHANG, CHIA-JUNG CHUNG, CHIA-JUNG LEE, KAI-CHUN PENGStudents: YI-CHAO CHEN, CHIH-HANG LIN, PO-HUA CHEN, WEN-HUI CHENG, HUIERH LEE, WOAN MEI JEAN, BO-NENG SYU, CHUN-HAO PAN, TA-YU LIU, HELENA LEE, CHUN-SAN TAI, YI-MIN DENG, HAO-HSIEN WANG, CHIA-CHING WU, PO-WEI CHIANG, YI-CHUN TSAI, YA-HSUAN LIN, TONG WENG SI , CALVIN HUE

AbstractThe majority of the previous iGEM projects focused on expressingcertain genes to achieve a specific task such as sensing a certainphenomenon. This year, we took a step forward in building a systemthat is capable of regulating multiple genes in E.coil. The system, so-called E.colightuner, is built by three regulated-systems which usednoninvasive factors such as red light and temperature to createdifferent conditions under which E. coli can express different genes.We also introduced a new sRNA regulated-system that hasn't beenwidely applied in iGEM before.

Red Light-regulated systemFor cph1 to sense light in E. coli,

it requires phycocyanobilin (PCB).We therefore introduced twoPCB-biosynthesis genes, ho1 andpcya from Synechocystis, con-verting heme into PCB.

With PCB, cph1 serves as a red-light absorbing chromophore thatis inactivated under red light andactivated without red light . Fig1.The light receptor cph8 is composed of

cph1(pink) and envZ-ompR(maroon).

Based on this sensor, wedesigned a BioBrick called redpromoter — Pred which containsPlac and LacI downstream ofompC promoter. In an oppositeway to cph1, Pred is activated byred light and deactivated withoutred light.

Fig3. Comparing the efficiency of Pred under redlight and dark. The data indicates that our Pred

do actually work.

Fig2. Red promoter

Temperature-regulated systemThe secondary structure of RNA thermometer masks the SDsequence and prevents ribosome binding, therefore initiation oftranslation is forestalled. When the temperature reaches 37 °C, thebase-pairing nucleotides loose their binding forces and expose theSD sequence to ribosome. In this way, gene expression can beregulated on the RNA level by temperature.

Fig4 . The mechanism of temperature-regulated system.

The normalized expression(fluorescence expression/ ODvalue) under 37 oC is higherthan the expression under 25 oCby approximately 6 folds.

Fig5. The functional test of 37 oC RBS.

A non-coding sRNA would recognize a special sequence called the sRNA-binding site.Once the sRNA binds to the sRNA-binding site, it would hinder ribosomes frombinding and initiating translation. Here, we designed two artificial sRNAs, eachmodified from different paper. We also designed a RBS specifically targeted by oursRNA. By adding this RBS to the upstream of any desired gene, the gene can beregulated by our sRNA. Unlike the regulated-systems popularly used in iGEMprojects, sRNA regulated-system seems more efficient.

Fig6. Inhibition mechanism of sRNA. Fig7. Artificial sRNA.

Fig10. As time goes, the normalized fluorescenceexpression approaches approximately five hundred AU,meaning that sRNA can effectively suppress geneexpression.

sRNA-regulated system

Fig8. Compare of the strength ofrRBS(K1017202) with other RBS(B0030,B0032, B0034).

Fig9. As the inhibition works under RNA level, the energy wasted in E. coli is less than other system, causing only harmless effect on E. coli growth.

By replacing the fluorescent proteins with plant hormones, we can offer multiplehormones secreted by E. coli in different growing stages to help the plants grow.

Fig13. The above is our device to culture plants.

Application

Fig14. Add different frequencies of light or use different intensity of red light and temperature to regulate more genes.

Human PracticeWe hosted a four-day iGEM conference which weinvited NYMU, NTU, SJTU, ZJU, and HKU toparticipate and we also held Bio-camp for highschool students, extending synthetic biology. We’reeager to share our ideas to other brilliant teams, andwe learned from them as well.

Red Light Induced Circuit

Dark Response Circuit

Mechanism of E.colightuner

25 °C

37 °C

RFP expression

GFP expression

25°C YFP expression

37 °C BFP expression

Red light

Red light

Dark

Dark

Fig11. The overall pathway of our project.

Fig12. Different gene expressions of E.colightuner.

New BioBrickrRBS: sRNA target RBS -K1017202sRNA: a functional sequence inhibit promoter -K1017404

Correct cph8 registry part -K1017301Increase the convenience of red light sensing system

Improvement

Contributions

生物科技學院