Cockroach terminator

The SZU-China team won the golden medal for best manufacturing project and was the second runner up for the Grand prize. Web page of SZU-China team is: http://2018.igem.org/Team:SZU-China<ref>[1]</ref>.

Introduction

Cockroaches are among the most common household pests, they harbor in damp and unsanitary places such as sewers, garbage disposals, kitchens, and bathroom, feed on human’s and pet’s food. Cockroaches have been implicated in the transmission of several pathogenic organisms such as E.coli and Salmonella enteritidis which can cause diarrhea, pneumonia and so on. <ref name=[2]>Mpuchane S, Matsheka IM, Gashe BA, Allotey J, Murindamombe G, Mrema N. Microbiological studies of cockroaches from three localities in Gaborone, Botswana. Afr J Food Nutr Sci. 2006;6:56–59</ref> Currently, there are two ways to control the population of cockroaches, physical and chemical methods. However, a chemical method like foggers, boric acid, and gel bait may cause environmental toxicity and the development of resistance in cockroaches. A physical method such as “cockroach house”, only trap few cockroaches. Neither chemical nor physical method can’t achieve the goal of environment-friendly and efficient control. <ref>[3]</ref>

Chassis and parts

The SZU-China team choose a kind of entomogenous fungi, Metarhizium anisopliae, as biological chassis. It is considered a safe and prospective choice for causing disease in insects only. <ref>[4]</ref> Its infection process is divided into the following steps:

1. spore attachment

2. penetration through the body wall

3. colonization in vivo

4. killing the host

5. re-sporulation.

It is a natural insecticide, and by genetically-enhancing it, we can make better use of it. But its lethality is still needed to be improved, which limits it's widespread used. They construct a system to enhance its virulence. Their system contains three parts: HsbA, BbChit, and MCL1. These three genes work sequentially during infecting cockroach, promote adhesion, penetration, and immune-avoidance respectively. HsbA- The HsbA from Beauveria bassiana (the entomopathogenic fungi, used as a biological insecticide) encodes a membrane surface hydrophobic protein A, located on the surface of fungus. This protein works like “glue” by forming a hydrophobic bond between spores and the waxy epicuticle of their host. <ref>[5]</ref>

BbChit- Cockroaches’ body wall is composed of protein, chitin, and lipids. BbChit, which comes from Beauveria bassiana, encodes chitinase which can hydrolize chitin to penetrate the host. Thereby, fungus can enter into hemolymph. <ref>[6]</ref>

MCL1-It encodes a collagen-like protein, which can combine with β-glucan. β-glucan is distributed on the cell wall surface of fungus and is the recognization site for insect hemocytes to combine and clear invader. MCL1 acts like putting an “invisible cloak” on the fungus, so that fungus can evade being recognized by the host immune system. Cockroaches have powerful immune system. If there is no effective response, their chassis won't be recognized and killed by host hemocytes. The MCL1 gene from Metarhizium Robertsii can encode collagen-like protein, which binds and masks β-1,3-glucan (an antigen that can be recognized by hemocytes), thus Metarhizium anisopliae can escape the immune response of cockroaches. <ref>[7]</ref>

Safety mechanism

They designed a suicide switch for their system. It consists of tryptophan attenuator and MazF (venom protein). Tryptophan attenuator will be switched on in high Trp concentration, while it will be switched off on the contrary. To make MazF expressed in chassis, they added a Kozak sequence in front of these two parts. They create a high Tryptophan concentration environment while culturing the fungus, and average concentration of Trp inside cockroach hemolymph is about 0.15%. In those environments, the tryptophan attenuator forms a hairpin structure that stop transcription ahead of MazF, so that fungus can stay alive. If the fungus live in the low tryptophan environment , the suicide switch will be turned on and the MazF starts to express, which leads to death of fungus. <ref>[8]</ref>

Construction of plasmid vector

They used the fungal plasmid pBC for their project, which can propagate in Metarhizium anisopliae. The vector contains the following parts:

PgpD is the constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans; TtrpC is a tryptophan terminator from Aspergillus nidulans; HsbA is encoded by the gene HsbA from Beauveria bassiana; BbChit is encoded by the gene Bbchit from Beauveria bassiana; MCL1_Metarhizium robertsii and Tryptophan-MazF-suicide switch, induces apoptosis in the absence of tryptophan.

Inside the vectors, there are four gene pathways. PgpdA promoter starts the transcription of HsbA, Bbchit, MCL1, Ttyptophan-MazF. TtrpC terminator terminates the transcription. It is well to be reminded that the pathway, PgpdA-Ttyptophan-MazF-TtrpC has a special switch, which induces apoptosis in the absence of tryptophan.

Results

Adhesion:HsbA

In this part, a strong promoter, PgpdA allows the HsbA protein to be expressed without induction. In the HsbA macro verification protocol, they could compare whether there was any change of the position and number of spores in the observing area. The wild-type Metarhizium anisopliae groups had the adherence rate in average of 26.7%. The Metarhizium anisopliae HsbA transformant groups had the adherence rate in average of 97.7%. In conclusion, this result confirmed that Metarhizium anisopliae HsbA transformant certainly enhanced the capacity of adhesion.

Penetration:BbChit

In order to verify the function of Bbchit, they improved Kan Zhuo's chitin transparent circle method for verification. They stained the czapek solid medium without chitin colloids in red with 0.1% Congo red dye solution and then inoculated wild-type and transformed Metarhizium.Then they compared the size of the colony and the transparent circle between wild type and transformant. The size measured is the diameter of the chitin transparent ring (R2) and colony(R1). The ratio of the diameters showed as R2/R1.The conclusion is that the chitinase activity of transformed Metarhizium anisopliae is enhanced by 1.3 times.

Immune avoidance:MCL1

In this part, PgpdA is a strong promoter that allows MCL1 to be expressed without induction. Change of nodules formed of hemocytes in cockroach hemolymph after injecting hyphae homogenate. At the time point of 0.5h, 1h and 8h, the nodules caused by M.anasopliae is significantly higher than transformants´ which means immune-avoidance occurs in genetically enhanced M.anasopliae.

Suicide switch

In order to confirm the limited concentration of tryptophan, they did a macro experiment. They put Metarhizium in an L-Tryptophan concentration gradient Petrie dishes from 0.05% to 0.14% with solid and liquid czapek. It was seen that the Metarhizium can stay alive at the L-Trp concentration of 0.09% or higher while it could not grow well or die at a lower concentration. They could also see that Metarhizium could not survive without L-Tryptophan.

GreenGround trapbox

Using all four parts they designed a trap box named GreenGround to bring their idea into real world. They mixed spores, banana powder and oil together to form emulsifiable powder, which is applied to non-woven fabrics for use. They developed it in three generations.

First generation

At first, they used oil solution to contain Metarhizium anisopliae. One area was a hollow cube to contain baits for cockroaches. There were breathable holes on top of it to make sure the smell of the baits got out. There were four entrances which allowed the cockroaches to enter. When cockroaches got in, they would get in contact with Metarhizium anisopliae which would kill them. And the cockroaches could also get out and infect other cockroaches by contacting them. After the human practice in Chongqing, they knew that cockroaches will be likely to climb gentle slope instead of entrances it.

Second generation

They decided to use Metarhizium anisopliae emulsifiable powder on nonwoven fabric instead of oil solution. This time they mixed the baits with Metarhizium anisopliae emulsifiable powder and brush them on nonwoven fabric. They made a box which formed a cleft for the cockroaches to climb in through the gentle slope. In the middle were 5 magnet cubes, which could adjust the height of the cleft according to the actual size of the cockroach. They found out that magnets are not stable enough and that acrylic has its own limitations.

Final generation

After they found acrylic can no longer fit their needs, they change the material into polypropylene. They also mix baits with Metarhizium anisopliae emulsifiable powder on polypropylene surface. They screw the lid down through the cylinder to form cleft which is adjustable. The cockroaches can climb through the slope and get in contact with Metarhizium anisopliae which will lead to death.

Conclusion

GreenGround trap box is safe and easy to use. It does not cause environmental toxicity and the development of resistance in cockroaches. Their improved M. anisopliae has better adhesion rate, is more successful in penetrating the host and can avoid immune system of the host, comparing to WT M. anisopliae. They wanted to make their product avalliable for the public since lots of poor neighborhoods in China have problem with cockroaches and other insects. They suceed in that by improving they trap box to maximize its function and to make it cheap. They also put their GreenGround box on popular app so they can get a feedback from public.

Literature

[1] http://2018.igem.org/Team:SZU-China

[2] Mpuchane S, Matsheka IM, Gashe BA, Allotey J, Murindamombe G, Mrema N. Microbiological studies of cockroaches from three localities in Gaborone, Botswana. Afr J Food Nutr Sci. 2006;6:56–59

[3] Wu X, Appel AG.J Econ Entomol. 2017 Jun 1;110(3):1203-1209

[4] Wang C, Lü Dingding, Li Lin. Study on pathogenicity and degradation mechanism of entomogenous fungi [C]// Chinese Society of Fungal Sciences Academic Symposium. 2008

[5] Ye Zhang, Zhongren Lei, Haihong Wang, Jiqing Zhan. Prokaryotic expression and immunolocalization Beauveria bassiana HsbA protein [J] Chinese Agricultural Sciences, 2013,46 (21): 4534-4541

[6] Liu Zhihui, Chen Shouwen, Guo Zhihong, et al. Correlation between extracellular protease and chitinase activity of Beauveria bassiana and virulence to Asian corn borer[J]. Journal of Huazhong Agricultural University, 2005, 24(4) :364-368

[7] Wang C, St Leger R J. A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses.[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(17):6647-6652

[8] Sowa S M, Keeley L L. Free amino acids in the hemolymph of the cockroach, Blaberus discoidalis[J]. Comparative Biochemistry & Physiology Part A Physiology, 1996, 113(2):131