Team:NYMU-Taipei/Project/Inhibition/Sensor

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National Yang Ming University


Contents

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 [http://parts.igem.org/Part:BBa_J23102 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 ([http://parts.igem.org/Part:BBa_K362001 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.

[http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-12951&orgids=ECOLI HemHp]([http://parts.igem.org/Part:BBa_K1104202 K1104202])
[http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-3104&detail-level=1 TrxCp]([http://parts.igem.org/Part:BBa_K1104201 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 [http://parts.igem.org/Part:BBa_K362001 Part:BBa_K362001](ahpC)and [http://parts.igem.org/Part:BBa_K362005 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 [http://parts.igem.org/Part:BBa_K554000 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 [http://ecocyc.org/ Ecocyc]and tested promoters: [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-12951&orgids=ECOLI HemHp] and [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU0-3104&detail-level=1 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: [http://parts.igem.org/Part:BBa_K1104202 Part:BBa_K1104202]、[http://parts.igem.org/Part:BBa_K1104201 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

OxyR-included circuit(Device1+Device2)
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 ([http://parts.igem.org/Part:BBa_E0840 Part:BBa_E0840]). Device1 in order to enhance the effect of ROS on E. coli is added ahead: a constitutive promoter([http://parts.igem.org/Part:BBa_J23102 Part:BBa_J23102]), RBS([http://parts.igem.org/Part:BBa_B0034 Part:BBa_B0034]), activator OxyR([http://parts.igem.org/Part:BBa_K1104200 Part:BBa_K1104200]),and double terminator [http://parts.igem.org/Part:BBa_B0015 Part:BBa_B0015].
Device1
a sensor (OxyR-induced promoter, including TrxCp, HemHp, sufA, AhpCp(mutated), AhpCp2D1, AhpCp2, AhpCpD1, AhpCp1, DsbGp) + reporter ([http://parts.igem.org/Part:BBa_E0840 Part:BBa_E0840])
Device2
a constitutive promoter([http://parts.igem.org/Part:BBa_J23102 Part:BBa_J23102])+ RBS([http://parts.igem.org/Part:BBa_B0034 Part:BBa_B0034])+ activator OxyR([http://parts.igem.org/Part:BBa_K1104200 Part:BBa_K1104200])+ double terminator [http://parts.igem.org/Part:BBa_B0015 Part:BBa_B0015]
Parts
[http://parts.igem.org/Part:BBa_J23102 Part:BBa_J23102]
RBS
([http://parts.igem.org/Part:BBa_B0034 Part:BBa_B0034])
OxyR coding
([http://parts.igem.org/Part:BBa_K1104200 Part:BBa_K1104200])
[http://parts.igem.org/Part:BBa_B0015 Part:BBa_B0015]
[http://parts.igem.org/Part:BBa_E0840 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
2.HemHp
([http://parts.igem.org/Part:BBa_K1104202 Part:BBa_K1104202])
  • Promoters on PartRegistry
3.sufA
([http://parts.igem.org/Part:BBa_K362005 Part:BBa_K362005])
  • Improved-Promoters from [http://parts.igem.org/Part:BBa_K362001 ahpC(Part:BBa_K362001)]
5.AhpCp2D1
([http://parts.igem.org/Part:BBa_K1104204 Part:BBa_K1104204])
6.AhpCp2
([http://parts.igem.org/Part:BBa_K1104205 Part:BBa_K1104205])
7.AhpCpD1
([http://parts.igem.org/Part:BBa_K1104206 Part:BBa_K1104206])
8.AhpCp1
([http://parts.igem.org/Part:BBa_K1104207 Part:BBa_K1104207])
9.DsbGp
([http://parts.igem.org/Part:BBa_K1104207 Part:BBa_K1104207])

SoxR-included circuit

OxyR-included circuit(Device1+Device2)
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 reporter[http://parts.igem.org/Part:BBa_E0840 Part:BBa_E0840] downstream the promoter. It is also composed of 2 devices:One is a sensor SoxS([http://parts.igem.org/Part:BBa_K554003 Part:BBa_K554003]) plus reporter ([http://parts.igem.org/Part:BBa_E0840 Part:BBa_E0840]). The other device is a constitutive promoter([http://parts.igem.org/Part:BBa_J23102 Part:BBa_J23102]), RBS([http://parts.igem.org/Part:BBa_B0034 Part:BBa_B0034]), activator SoxR([http://parts.igem.org/Part:BBa_K554003 Part:BBa_K554003]),and double terminator [http://parts.igem.org/Part:BBa_B0015 Part:BBa_B0015].
Device2
Parts
SoxR
([http://parts.igem.org/Part:BBa_K554003 Part:BBa_K554003])
[http://parts.igem.org/Part:BBa_B0015 Part:BBa_B0015]
SoxS
([http://parts.igem.org/Part:BBa_K554000 Part:BBa_K554000])
[http://parts.igem.org/Part:BBa_E0840 Part:BBa_E0840]

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 ([http://parts.igem.org/Part:BBa_K1104201:Experience Part:BBa_K1104201:Experience])
  2. HemHp ([http://parts.igem.org/Part:BBa_K1104202:Experience Part:BBa_K1104202:Experience])
  3. sufA ([http://parts.igem.org/Part:BBa_K1104205:Experience Part:BBa_K1104205:Experience])
  4. ahpC(mutated)([http://parts.igem.org/Part:BBa_K1104203:Experience Part:BBa_K1104203:Experience])
  5. AhpCp2D1([http://parts.igem.org/Part:BBa_K1104204:Experience Part:BBa_K1104204:Experience])
  6. AhpCp2([http://parts.igem.org/Part:BBa_K1104205:Experience Part:BBa_K1104205:Experience])
  7. AhpCpD1([http://parts.igem.org/Part:BBa_K1104206:Experience Part:BBa_K1104206:Experience])
  8. AhpCp1([http://parts.igem.org/Part:BBa_K1104207:Experience Part:BBa_K1104207:Experience])
  9. DsbGp([http://parts.igem.org/Part:BBa_K1104208:Experience 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([http://parts.igem.org/Part:BBa_K554000:Experience Part:BBa_K554000:Experience]).

Part Improvement

We improve the function of a BioBrick Part: ahpC promoter([http://parts.igem.org/Part:BBa_K362001 Part:BBa_K362001])designed by 2010 KIT-Tokyo team into four versions.On PartRegistry, the complex part according to [http://ecocyc.org/ECOLI/new-image?object=EG11384 Ecocyc]composition 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.

ahpC([http://parts.igem.org/Part:BBa_K362001 Part:BBa_K362001])
AhpCp(mutated)[http://parts.igem.org/Part:BBa_K1104203 Part:BBa_K1104203]
We succesfully mutated the Pst1 site (ctgcag->ctacag) of K362001.
AhpCp2D1([http://parts.igem.org/Part:BBa_K1104204 Part:BBa_K1104204])
We delete the dsbG coding and following sequence next to DsbGp reverse primer, making the functional sequence shorter, so that PCR can be more easy and efficient.
AhpCpD1([http://parts.igem.org/Part:BBa_K1104206 Part:BBa_K1104206])
We designed a dual-side hybrid promoter, including AhpCp1 and DsbGp(reverse), and two binding sites for OxyR. OxyR binding to TFBS leads to dual-side transcription.

Because ahpC([http://parts.igem.org/Part:BBa_K362001 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.

AhpCp1
[http://parts.igem.org/Part:BBa_K1104207 Part:BBa_K1104207]
AhpCp2
[http://parts.igem.org/Part:BBa_K1104205 Part:BBa_K1104205]
DsbGp
([http://parts.igem.org/Part:BBa_K1104207 Part:BBa_K1104207])

Discussion

As the results shows, we successfully improved AhpC promoter([http://parts.igem.org/Part:BBa_K362001 Part:BBa_K362001]). And the results and disscussion on PartRegistry are linked below:

  1. AhpCp(mutated)([http://parts.igem.org/Part:BBa_K1104203:Experience Part:BBa_K1104203:Experience])
  2. AhpCp2D1([http://parts.igem.org/Part:BBa_K1104204:Experience Part:BBa_K1104204:Experience])
  3. AhpCp2([http://parts.igem.org/Part:BBa_K1104205:Experience Part:BBa_K1104205:Experience])
  4. AhpCpD1([http://parts.igem.org/Part:BBa_K1104206:Experience Part:BBa_K1104206:Experience])
  5. AhpCp1([http://parts.igem.org/Part:BBa_K1104207:Experience Part:BBa_K1104207:Experience])
  6. DsbGp([http://parts.igem.org/Part:BBa_K1104208:Experience 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[http://www.nature.com/nrm/journal/v8/n10/fig_tab/nrm2256_F1.html]
  3. Ryu, S. E. (June 01, 2012). Structural mechanism of disulphide bond-mediated redox switches. Journal of Biochemistry, 151, 6, 579-588.[http://jb.oxfordjournals.org/content/early/2012/05/02/jb.mvs046.full.pdf]
  4. [http://ecocyc.org/ Ecocyc]
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