Applet Human Practices Experiment Project Team Awards

Design

Background

As the background we described, to solve the problem that cockroaches’ flooding in home environment, we use the transformation of the chafer and the green deadlock to achieve the effect of killing the cockroaches. In order to ensure the killing ability of Metarhizium anisopliae to the cockroaches, we improved in three ways: the adhesion, penetration, and survivability.

Which chassis to choose and why?

The chassis organism we chose was Metarhizium anisopliae. This is an entomogenous fungus that infects more than 200 species of insects, most of which are agricultural pests also includes cockroaches^[1]. Its infection process is divided into the following steps: spore attachment -- penetration of the body wall -- in vivo colonization -- killing the host -- re-spore formation. It is a natural insecticide tool, and by transforming it, we can make better use of it.

How to achieve?

We designed the system to transform Metarhizium anisopliae from three parts: HsbA, Bbchit and MCL1.

HsbA: The HsbA from Beauveria bassiana encodes a kind of membrane surface hydrophobic protein which helps our spores adhere to the wax on the cockroach body surface^[2]. Moreover, with the overexpression of HsbA, our spores can more effectively adhere to the cockroach. Then it will follow as spores’ germination, germinal tube, appressorium, and the next penetrating process.

Bbchit: Cockroaches’ body wall composed of protein, chitin, and lipids. Bbchit is a chitinase gene which comes from Beauveria bassiana. It can " bore a hole" by decomposing chitin on the cockroaches. Thereby allowing the hyphae to enter the cockroaches’ body^[3].

MCL1: Cockroaches’ immune system is powerful. If there is no effective response, our chassis will be recognized and killed by blood cells easier. The MCL1 gene from Metarhizium Robertsii can encode a protein similar to host collagen, which binds to the surface of our chassis and masks β-1,3-glucan (an antigen that can be recognized by blood cells), thus enabling Metarhizium anisopliae escapes the immune response of cockroaches^[4].

How to ensure safety?

We built a safety mechanism for our system. It consists of a tryptophan attenuator and a MazF venom protein both come from the prokaryotic system. To make it expressed in our chassis organisms, we added a Kozak sequence to the front of these two parts. We put a suitable concentration of Tryptophan while we are culturing the fungi, and the average concentration of Trp inside cockroach is about 0.15%^[5]. In both of the environment, the Tryptophan attenuator makes a difference that stops the translation before MazF, and then the fungi can keep alive. If the fungi abscise the environment we mention above, the Tryptophan attenuator would not work and the MazF expresses to make them die quickly.

References

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

[2] 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.

[3] 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.

[4] 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.