View source for SURF 2024: Bioengineering toolkit development for genetic alterations in the entomopathogenic nematode symbiont Xenorhabdus griffiniae

'''[[SURF 2024|SURF 2024]] project description''' __NOTOC__
* Mentor: Richard Murray
* Co-mentor: Elin Larsson

== Background ==

Nematodes are key players in the soil environment, where they feed on bacteria, fungi, insects and plant roots. Therefore, they affect decomposition of organic matter, cycling of nutrients  and population size and distribution of other living organisms. Entomopathogenic nematodes (EPN, insect-parasitic) and their symbiotic bacteria are crucial to soil ecology (1,2). The bacterium Xenorhabdus griffiniae is the core symbiont that associates with Steinernema hermaphroditum, an EPN that seeks out and infects insects. The symbiotic pair kills the insect and uses the nutrient from the insect cadaver for reproduction (Fig. 1) (3). To smoothly transition and adapt to complex host environments including the nematode intestine, insect immune system, and decaying insect cadaver, Xenorhabdus bacteria must encode molecular mechanisms to sense the signals from each microenvironment, and regulate its gene expression accordingly. Therefore, it is crucial to characterize gene expression patterns within its relevant ecological niche to better understand and manipulate the bacterial behaviors, such as colonization of nematodes, virulence towards insects, and production of attractants, repellents, and nematicidal metabolites.


[[File:lifecycle.png|500px|thumb|right|Figure 1: The lifecycle of Steinernema hermaphroditum (blue) and its symbiont Xenorhabdus griffiniae (pink) harbored in the nematode receptacle during the host seeking phase.]]


In order to achieve this goal, tool development in both partners is necessary. Although basic genetic tools, such as conjugation and transformation, are available in Xenorhabdus bacteria (4), systematic and efficient toolkits in bioengineering are lacking, which has limited the basic research and application of these species. Current synthetic biology tools have been proven powerful in bioengineering, but are largely limited to bacterial model organisms like E. coli and B. subtilis, and the yeast S.cerevisiae. To understand ecologically relevant interactions among species, the same tools need to become available in non- and emerging model organisms. In this project, we are focusing on systematically establishing a part library (Murray lab expertise) in X. griffiniae to expand the capacity of bioengineering in this species. As a proof of concept, we will adapt a small-scale part library from E. coli in X. griffiniae for in vitro screening of bacterial gene expression. Relevant candidate bacterial strains will be tested further in vivo using its natural mutualistic host nematode (Cao lab expertise). The proposed effort in tool development will be crucial to elucidating signaling between the nematode and bacteria in this partnership and potentially expand its application to soil microbe engineering.

== Research overview ==

The SURF student will use molecular biology techniques to generate a genetic part library, a collection of promoters, ribosome binding sites, fluorescent proteins with different properties, for X. griffiniae, largely using the pre-existing part library available in the lab for E. coli. Even though genetic parts often function across species, there are many examples where they function differently or not at all. Therefore, it is important to characterize a range of the aforementioned parts in the genetic background of the species of interest, to be able to predictably build different variants of genetic constructs.


=== Aim 1: Creating a genetic part library for X. griffiniae ===

The first aim of the project is to generate a small part library that is compatible with the 3G cloning pipeline (5). First, Xenorhabdus vectors will be modified to fit into the E. coli cloning pipeline. The vectors can then be used to build a range of constructs using the parts already available in the Murray lab part library .
The plasmids will then be integrated on the X. griffiniae genome using conjugation (or trying to develop electroporation if this is preferable).

=== Aim 2: Characterization of parts in vitro ===

After creating a library of parts, these can be characterized in a plate reader assay. The strains will be grown in minimal medium (+/- inducers where appropriate). The functionality and rank order of the parts in this species can then be determined.

=== Aim 3: Characterization of parts in vivo ===

Last of all we want to see if the in vitro behavior can translate to an ecologically relevant context. To do this, a genetic construct will be designed for bacteria colonizing the worm receptacle. The in vitro performance can be determined using fluorescence microscopy or worm viability depending on the intended construct output.

'''SURF student qualifications:'''
* Prerequisite coursework: Bi1x, Bi8/9 (or similar introductory biology course that provides a basic foundation in molecular biology)
*Preferred: Familiarity with synthetic biology/circuit design
*Basic familiarity with coding in Python



'''References:'''

1.	Abd-Elgawad MMM. spp.: An Overview of the Useful Facets of Mutualistic Bacteria of Entomopathogenic Nematodes. Life [Internet]. 2022 Aug 31;12(9). Available from: http://dx.doi.org/10.3390/life12091360

2.	Kenney E, Eleftherianos I. Entomopathogenic and plant pathogenic nematodes as opposing forces in agriculture. Int J Parasitol. 2016 Jan;46(1):13–9.

3.	Cao M, Schwartz HT, Tan CH, Sternberg PW. The entomopathogenic nematode Steinernema hermaphroditum is a self-fertilizing hermaphrodite and a genetically tractable system for the study of parasitic and mutualistic symbiosis. Genetics [Internet]. 2022 Jan 4;220(1). Available from: http://dx.doi.org/10.1093/genetics/iyab170

4.	Alani OS, Cao M, Goodrich-Blair H, Heppert JK. Conjugation and transposon mutagenesis of HGB2511, the bacterial symbiont of the nematode (India). MicroPubl Biol [Internet]. 2023 Apr 25;2023. Available from: http://dx.doi.org/10.17912/micropub.biology.000772

5.	Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. ACS Synth Biol. 2018 May 18;7(5):1477–80.