National Yang Ming University


Sensing Nosema ceranae


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 added a new composition part: device:BBa_K1104250 consisting of a constitutive promoter Part:BBa_J23102 and a transcription factor: OxyR (Part:BBa_K1104200), which can activate ROS-sensing promoters, increasing the quantity of inactivated transcription factors in order to enhance the response of promoters. Additionally, RBS (Part:BBa_B0034) and double terminator Part:BBa_B0015. Actually, the addition of device:BBa_K1104250 remarkably enhanced the strength of promoter AhpCp2D1(Part:BBa_K1104204) in our experiment results.

We improved the function of a BioBrick Part: ahpC promoter (Part:BBa_K362001) originally designed by 2010 KIT-Tokyo team.


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 infects the midgut of bees, midgut cells of bees will secrete Reactive Oxygen Species (ROS). ROS have been shown to be toxic but also function as signalling molecules which can transcriptionally activate transcription factors: OxyR (Part:BBa_K1104200). We focus on ROS-induced promoters regulated by OxyR.

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

Activated transcription factor, OxyR, then binds to TFBS of promoters (sensor), as the pictures shown below:

We did a study about promoters that can sense OxyR. Several promoters on the 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.

We also got the promoter sequences taken from Ecocyc and 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 designed and sent them to PartRegistry as new parts: Part:BBa_K1104202Part:BBa_K1104201.


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

In order to enhance the strength and sensitivity of ROS-sensing promoters, we made E. coli continuously produce inactivated OxyR, so that ROS can affect promoters more easily.

OxyR-included circuit

OxyR-included circuit(Device1+Device2)

In OxyR-included circuit, Device2 is composed of sensor (OxyR-induced promoter, including TrxCp, HemHp, sufA, AhpCp1000, 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.

Putting Device1 device:BBa_K1104250 upstream to promoter AhpCp2D1(Part:BBa_K1104204) remarkably enhanced the strength of promoter AhpCp2D1(Part:BBa_K1104204) as our experiment results shows.

Device1 (Part:BBa_K1104250)
a constitutive promoter(Part:BBa_J23102)+ RBS(Part:BBa_B0034)+ activator OxyR(Part:BBa_K1104200)+ double terminator Part:BBa_B0015
a sensor (OxyR-induced promoter, including TrxCp, HemHp, sufA, AhpCp1000, AhpCp2D1, AhpCp2, AhpCpD1, AhpCp1, DsbGp) + reporter (Part:BBa_E0840)
OxyR coding
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


We tested Device2 of each OxyR-induced promoters, as well as Device1+Device2 of AhpCp2D1. And the results of OxyR-induced promoters testing on PartRegistry are linked as follows:

NYMU pp.png
Above shows the strengths of the basal level of various promoters that respond to reactive oxygen species with respect to BBa_J23101.
In the presence of Device 1, AhpCp2D1 shows 7 to 9 folds of strength (Error bars represent the 95% confidence interval)

Part Improvement

There is a Pst1 site in the red rectangle.

We improved the function of a BioBrick Part: ahpC promoter (Part:BBa_K362001) designed by 2010 KIT-Tokyo team into four versions. On the PartRegistry, the complex part according to Ecocyc composition contains part of DsbG coding, 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.

We succesfully mutated the Pst1 site (ctgcag->ctacag) of K362001.
We strimmed DsbG coding out of the original sequence, making the functional sequence shorter and more efficient.
We designed a dual-side hybrid promoter, including AhpCp1 and DsbGp(reverse), and two binding sites for OxyR. OxyR binding to TFBS leads to bidirectional transcription.

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


As the results show, we successfully improved ahpC promoter (Part:BBa_K362001). And the results and disscussion on the PartRegistry are linked as follows:

Above shows promoter testing of ahpC promoter Part:K362001) and its improvements, which suggests that AhpCpd1 and AhpCp1 show so weak promoter strength that we only focus on the change of promoter strength of the three promoters as following:


  • 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.
  • 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]
  • Ryu, S. E. (June 01, 2012). Structural mechanism of disulphide bond-mediated redox switches. Journal of Biochemistry, 151, 6, 579-588.[2]
  • Ecocyc.