Team:NYMU-Taipei/Project/Inhibition/Sensor

Contents 1 Introduction 2 Background 2.1 Nosema Ceranae Induces Oxidative Stress in the Midgut. 2.2 Literature Study of ROS-induced Promoters 3 Method 3.1 How Can Bee. coli Sense Nosema? 3.2 Circuit Design 3.2.1 OxyR-included circuit 3.2.1.1 Parts 3.2.1.2 OxyR-activated promoters 3.2.2 SoxR-included circuit 3.2.2.1 Parts 3.3 Disscussion 3.4 Part Improvement 3.5 Discussion 4 References

Introduction

• After Nosema ceranae enters the midgut of bees, we have to make E. coli sense this pathogen. E. coli with ROS-sensing promoters will respond to Reactive Oxygen Species (ROS) produced by infected midgut cells. The following circuit behind ROS-sensing promoters will be turned on and fight against N. cerenae.
• For the circuit design, we add a new part consisting of a constitutive promoter Part:BBa_J23102 and a transcription factor which can activate ROS-sensing promoters (such as OxyR or SoxR), increasing the quantity of inactivated transcription factors in order to enhance the response of promoters.
• We improve the function of a BioBrick Part: AhpC promoter (Part:BBa_K362001) originally designed by the 2010 KIT-Tokyo team.

Background

Nosema Ceranae Induces Oxidative Stress in the Midgut.

ROS(Reactive Oxygen Species)are efficient antimicrobial molecules. Genes involved in oxidation-reduction were significantly overrepresented in the gene set upregulated upon spore infection. In response to the infection, the increase of oxidoreduction in the midgut epithelia of bees infected by N. ceranae would therefore indicate an enhanced generation of ROS. It is suggested that ROS production is a general immune response in bees' midgut to microorganism infection. There are promoters in the genome of E. coli that can be regulated by ROS. After Nosema ceranae enter midgut of bee, the regulator of promoters will be activated by ROS, trigger the following circuit fighting against Nosema ceranae.

Literature Study of ROS-induced Promoters

Once N.cerenae infect the midgut of bee, midgut cells of bee will secret Reactive oxygen species (ROS). ROS have been shown to be toxic but also function as signalling molecules which can transcriptionally activate transcription factors: OxyR and SoxR. We focus on ROS-induced promoters regulated by either OxyR or SoxR.

OxyR Activation The OxyR transcription factor regulates the H2O2 response of E. coli. H2O2 oxidizes OxyR Cys199 to an R-SOH that reacts with Cys208 in an intramolecular disulphide bond.

SoxR Activation Oxidation of the SoxR [2Fe–2S] cluster by O2- causes a change in SoxR conformation, which alters the SoxR-bound operator DNA structure and results in gene activation.

Activated transcription factors, such as OxyR and SoxR, then bind to TFBS of promoters(sensor), as pictures show below.

HemHp(K1104202)

TrxCp(K1104201)

We did a study about promoters that can sense OxyR, or SoxR. Several promoters on part registry can be induced by ROS: OxyR-activated promoters such as Part:BBa_K362001(ahpC)and Part:BBa_K362005(sufA)were designed and used as favorite parts by 2010 KIT-Tokyo team. They can be controlled by OxyR. SoxR-activated part like Part:BBa_K554000(SoxS)was designed and used as favorite parts by 2011 UNICAMP-EMSE Brazil team, which can be controlled by SoxR.

We also got the sequence taken from Ecocycand tested promoters: HemHp and TrxCp. Both of them have dual binding site only interact with OxyR, cooperate with sigma factor 70, and have no E X S P cutting site. We have designedand sent them to partregistry as new parts: Part:BBa_K1104202、Part:BBa_K1104201.

Method

How Can Bee. coli Sense Nosema?

After Nosema ceranae infected midgut cells of bees, and , Bee. coli should sense the pathogen first before the following circuit(fighting against Nosema ceranae)is triggered. After Nosema ceranae infected midgut cells of bees, ROS(Reactive Oxygen Species) will be produced by midgut cells of bee and released to the cavity. As we can see, there are many kinds of promoters (and their transcription factors) responding to ROS naturally exist in E. coli MG1655 genome. We choose proper and strength enough ones from them, basing on reporting assays we made about promoetr testing. Besides, in order to enhance the strength, we designed an upstream device to enlarge the expression of the activators, so the sensitivity of Bee. coli to ROS will be sharper, and the probability of downstream circuit(fighting against Nosema ceranae) being turned on.

Circuit Design

There are two kinds of circuit: OxyR-included circuit and SoxR-included circuit, depending on the categories of promoters controled by different transcription factor. In order to enhance the strength and sensitivity of ROS-sensing promoters, we make E. coli continuously produce inactivated OxyR or SoxR, so that ROS can affect promoters more easily.

OxyR-included circuit

This is one kind of the two circuits--OxyR-included circuit. Device2 is composed of a sensor (OxyR-induced promoter, including TrxCp, HemHp, sufA, AhpCp(mutated), AhpCp2D1, AhpCp2, AhpCpD1, AhpCp1, DsbGp) plus reporter (Part:BBa_E0840). Device1 in order to enhance the effect of ROS on E. coli is added ahead: a constitutive promoter(Part:BBa_J23102), RBS(Part:BBa_B0034), activator OxyR(Part:BBa_K1104200),and double terminator Part:BBa_B0015.

Device1 a sensor (OxyR-induced promoter, including TrxCp, HemHp, sufA, AhpCp(mutated), AhpCp2D1, AhpCp2, AhpCpD1, AhpCp1, DsbGp) + reporter (Part:BBa_E0840)

Device2 a constitutive promoter(Part:BBa_J23102)+ RBS(Part:BBa_B0034)+ activator OxyR(Part:BBa_K1104200)+ double terminator Part:BBa_B0015

Parts

Part:BBa_J23102

RBS (Part:BBa_B0034)

OxyR coding (Part:BBa_K1104200)

Part:BBa_B0015

Part:BBa_E0840

OxyR-activated promoters

We searched for promoters that can be induced with ROS. Most of them activated by OxyR.

• Promoters on Ecocyc

• Promoters on PartRegistry

• Improved-Promoters from ahpC(Part:BBa_K362001)

SoxR-included circuit

This is the other kind of the two circuits--SoxR-included circuit. The deference is activator: in this circuit OxyR is replaced with SoxR. The construct is initially built by 2011 UNICAMP-EMSE Brazil team. We made some replacement to the original circuit: the constitutive promoter, double terminator, and a reporterPart:BBa_E0840 downstream the promoter. It is also composed of 2 devices:One is a sensor SoxS(Part:BBa_K554003) plus reporter (Part:BBa_E0840). The other device is a constitutive promoter(Part:BBa_J23102), RBS(Part:BBa_B0034), activator SoxR(Part:BBa_K554003),and double terminator Part:BBa_B0015.

Parts

Disscussion

We test only Device2, as well as Device1+Device2. And the results of OxyR-induced promoters testing on PartRegistry are linked below:

1. TrxCp (Part:BBa_K1104201:Experience)
2. HemHp (Part:BBa_K1104202:Experience)
3. sufA (Part:BBa_K1104205:Experience)
4. ahpC(mutated)(Part:BBa_K1104203:Experience)
5. AhpCp2D1(Part:BBa_K1104204:Experience)
6. AhpCp2(Part:BBa_K1104205:Experience)
7. AhpCpD1(Part:BBa_K1104206:Experience)
8. AhpCp1(Part:BBa_K1104207:Experience)
9. DsbGp(Part:BBa_K1104208:Experience)

The same as SoxR-induced promoter testing, we test Device2 and Device1+Device2. The construct is initially built by 2011 UNICAMP-EMSE Brazil team. We tested the Device with hydrogen peroxide instead of Paraquat(both of them are ROS). Besides, we made some replacement to the original circuit. The edited results of SoxR-induced promoters testing on PartRegistry: SoxS(Part:BBa_K554000:Experience).

Part Improvement

We improve the function of a BioBrick Part: ahpC promoter(Part:BBa_K362001)designed by 2010 KIT-Tokyo team into four versions.On PartRegistry, the complex part according to Ecocyccomposition contains hybrid promoters, shared TFBS (Transcription Factor Binding Site), and reverse promoter DsbGp. The intergenic region between DsbGp and AhpCp1 carries two binding sites for OxyR.

Because ahpC(Part:BBa_K362001) is a hybrid promoter containing AhpCp1, AhpCp2, and DsbGp promoter in its 1000bp, we create the new part and test it apart.

Discussion

As the results shows, we successfully improved AhpC promoter(Part:BBa_K362001). And the results and disscussion on PartRegistry are linked below:

1. AhpCp(mutated)(Part:BBa_K1104203:Experience)
2. AhpCp2D1(Part:BBa_K1104204:Experience)
3. AhpCp2(Part:BBa_K1104205:Experience)
4. AhpCpD1(Part:BBa_K1104206:Experience)
5. AhpCp1(Part:BBa_K1104207:Experience)
6. DsbGp(Part:BBa_K1104208:Experience)

References

1. Dussaubat, Claudia, Brunet, Jean-Luc, Higes, Mariano, Colbourne, John K., Lopez, Jacqueline, Choi, Jeong-Hyeon, Martín-Hernández, Raquel, ... Alaux, Cédric. (n.d.). Gut Pathology and Responses to the Microsporidium Nosema ceranae in the Honey Bee Apis mellifera. Public Library of Science.
2. D'Autréaux, B., & Toledano, M. B. (January 01, 2007). ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nature Reviews. Molecular Cell Biology, 8, 10, 813-24.2[1]
3. Ryu, S. E. (June 01, 2012). Structural mechanism of disulphide bond-mediated redox switches. Journal of Biochemistry, 151, 6, 579-588.[2]
4. Ecocyc