Murray Wiki - User contributions [en]

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-20T20:29:49Z

Mengcao:

'''[[SURF 2024|SURF 2024]] project description'''
[[File:Transgene modularity Library (1).jpeg|thumb|650px|right|Figure 1: Construction of transgene part library for ''Steinernema hermaphroditum'' (created in BioRender).]]
* Mentor: Mengyi Cao
* If you are interested in this topic, please check out our twin project (co-mentored by Elin Larsson):
{{SURF|2024|Bioengineering toolkit development for genetic alterations in the entomopathogenic nematode symbiont Xenorhabdus griffiniae}}

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with mutualistic symbiotic bacteria ''Xenorhabdus'' in a species-specific manner, and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they were discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no consistent transgenesis protocol in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the ''Steinernema'' species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be a powerful system in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit constructions (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in ''S. hermaphroditum (India)'', a recently isolated nematode strain with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon ''piggyBac'', which will be used to insert the constructs into the targeted sites in ''S. hermaphroditum'' genome. The modular part library will be stored in ''E. coli'' and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New ''E. coli'' Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-19T00:26:29Z

Mengcao:

'''[[SURF 2024|SURF 2024]] project description'''
[[File:Transgene modularity Library (1).jpeg|thumb|650px|right|Figure 1: Construction of transgene part library for ''Steinernema hermaphroditum'' (created in BioRender).]]
* Mentor: Mengyi Cao

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with mutualistic symbiotic bacteria ''Xenorhabdus'' in a species-specific manner, and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they were discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no consistent transgenesis protocol in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the ''Steinernema'' species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be a powerful system in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit constructions (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in ''S. hermaphroditum (India)'', a recently isolated nematode strain with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon ''piggyBac'', which will be used to insert the constructs into the targeted sites in ''S. hermaphroditum'' genome. The modular part library will be stored in ''E. coli'' and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New ''E. coli'' Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-19T00:11:50Z

Mengcao:

'''[[SURF 2024|SURF 2024]] project description'''
[[File:Transgene modularity Library (1).jpeg|thumb|650px|right|Figure 1: Construction of transgene part library for ''Steinernema hermaphroditum''.]]
* Mentor: Mengyi Cao

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with mutualistic symbiotic bacteria ''Xenorhabdus'' in a species-specific manner, and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they were discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no consistent transgenesis protocol in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the ''Steinernema'' species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be a powerful system in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit constructions (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in ''S. hermaphroditum (India)'', a recently isolated nematode strain with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon ''piggyBac'', which will be used to insert the constructs into the targeted sites in ''S. hermaphroditum'' genome. The modular part library will be stored in ''E. coli'' and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New ''E. coli'' Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).

File:Transgene modularity Library (1).jpeg

2023-12-19T00:03:48Z

Mengcao:

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-16T22:47:32Z

Mengcao: /* Aim 1: Cloning of modular parts. */

'''[[SURF 2024|SURF 2024]] project description'''
[[File:.jpg|thumb|500px|right|Figure 1: ]]
* Mentor: Mengyi Cao

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with mutualistic symbiotic bacteria ''Xenorhabdus'' in a species-specific manner, and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they were discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no consistent transgenesis protocol in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the ''Steinernema'' species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be a powerful system in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit constructions (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in ''S. hermaphroditum (India)'', a recently isolated nematode strain with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon ''piggyBac'', which will be used to insert the constructs into the targeted sites in ''S. hermaphroditum'' genome. The modular part library will be stored in ''E. coli'' and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New ''E. coli'' Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-16T22:46:34Z

Mengcao: /* References: */

'''[[SURF 2024|SURF 2024]] project description'''
[[File:.jpg|thumb|500px|right|Figure 1: ]]
* Mentor: Mengyi Cao

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with mutualistic symbiotic bacteria ''Xenorhabdus'' in a species-specific manner, and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they were discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no consistent transgenesis protocol in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the ''Steinernema'' species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be a powerful system in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit constructions (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in ''S. hermaphroditum (India)'', a recently isolated nematode strain with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon piggyBac, which will be used to insert the constructs into the targeted sites in ''S. hermaphroditum'' genome. The modular part library will be stored in E. coli and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New ''E. coli'' Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-16T22:38:24Z

Mengcao: /* Project Overview: */

'''[[SURF 2024|SURF 2024]] project description'''
[[File:.jpg|thumb|500px|right|Figure 1: ]]
* Mentor: Mengyi Cao

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with mutualistic symbiotic bacteria ''Xenorhabdus'' in a species-specific manner, and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they were discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no consistent transgenesis protocol in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the ''Steinernema'' species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be a powerful system in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit constructions (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in ''S. hermaphroditum (India)'', a recently isolated nematode strain with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon piggyBac, which will be used to insert the constructs into the targeted sites in ''S. hermaphroditum'' genome. The modular part library will be stored in E. coli and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-16T22:37:01Z

Mengcao: /* Project Overview: */

'''[[SURF 2024|SURF 2024]] project description'''
[[File:.jpg|thumb|500px|right|Figure 1: ]]
* Mentor: Mengyi Cao

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with mutualistic symbiotic bacteria ''Xenorhabdus'' in a species-specific manner, and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they were discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no transgenesis protocols in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the ''Steinernema'' species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be powerful in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit constructions (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in ''S. hermaphroditum (India)'', a recently isolated nematode strain with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon piggyBac, which will be used to insert the constructs into the targeted sites in ''S. hermaphroditum'' genome. The modular part library will be stored in E. coli and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-16T22:36:29Z

Mengcao: /* Project Overview: */

'''[[SURF 2024|SURF 2024]] project description'''
[[File:.jpg|thumb|500px|right|Figure 1: ]]
* Mentor: Mengyi Cao

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with mutualistic symbiotic bacteria ''Xenorhabdus'' in a species-specific manner, and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they are discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no transgenesis protocols in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the ''Steinernema'' species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be powerful in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit constructions (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in ''S. hermaphroditum (India)'', a recently isolated nematode strain with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon piggyBac, which will be used to insert the constructs into the targeted sites in ''S. hermaphroditum'' genome. The modular part library will be stored in E. coli and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).

SURF 2024

2023-12-16T01:16:24Z

Mengcao: /* List of available projects */

{{righttoc}}
This page is intended for students interested in working on SURF projects in the Summer of 2024. It contains information about how to apply for a SURF project in my group along with a list of project areas.

'''Note:''' Projects will be posted here starting after finals week and up to the start of classes. Please check back after that time for more information.

=== Applying for a SURF project in my group ===

Because I get many students interested in doing SURFs in my group and because we have several projects available, we use the first few weeks in January to sort out who we will work with in writing proposals. We only submit one proposal per project area and so we often can't accommodate everyone who wants to work in my group over the summer.

# A list of SURF project descriptions is given in the table below. Due to the number of SURF projects that we support, we are only able to support students who select from among these projects. Please make sure to read the project descriptions, required skills (if any) and skim a few of the listed references before contacting me about doing a SURF project.
# Students interested in writing proposals for SURF projects should contact me via e-mail by 10 Jan (Wed) and provide the following information:
#* A list of up to three SURF projects from the list below that you are interested in working on
#* A one page resume listing relevant experience and coursework
#* If you are not a Caltech student, I will also need the following additional information:
#** An unofficial copy of your academic transcript
#** Names of two faculty members at your current institution that I can contact for a reference
# Starting on 11 January, I will go through all applications and work with my group to identify who is a possible fit for each project. We will then contact you and ask for you to meet (or talk with) possible co-mentors so that we can eventually work out who we will work with in writing up a proposal.
# We hope to make final decisions on projects by about 1 Jan, at which point we will start working with students on writing up proposals.
# All applications should go through the normal SURF application process, described at www.surf.caltech.edu. SURF applications are due on ~22 Feb.
# If you are selected for a SURF, please be aware of the following information
#* All SURF projects in my group will start on 18 Jun (Tue). If you can't start on that date, please make sure that you indicate this when you contact me
#* All SURF projects are for a minimum of 10 weeks, although I usually recommend that you try to stay for 12 weeks if possible. It's hard to complete a project in just 10 weeks and spending a few extra weeks can greatly improve the project.
#* All SURF students in my group will be expected to devote full-time effort to their SURF project, so you cannot have a second job in addition to your SURF.
#* Additional information on SURF available here: https://sfp.caltech.edu/undergraduate-research/programs/surf

=== List of available projects ===

Projects will be posted as they come available. I recommend waiting until near the deadline submission before submitting your project preferences.

{| border=1 width=100%
|-
| '''Title''' || '''Grant/Project''' || '''Co-Mentors''' || '''Comments'''
|-
| {{SURF|2024|Task-Relevant Metrics for Perception}}
| TBD
| Apurva Badithela
|-
| {{SURF|2024|Hierarchical Testing for Safety-Critical Autonomous Systems}}
| TBD
| Apurva Badithela
|
|-
| {{SURF|2024|Establish synthetic biology toolkits for Steinernema nematode transgene expression}}
| Carnegie Institution for Science
| TBD
| Mentor: Mengyi Cao (PI)
|-
| {{SURF|2024|Bioengineering toolkit development for genetic alterations in the entomopathogenic nematode symbiont Xenorhabdus griffiniae}}
| TBD
| Elin Larsson
|
|-
| {{SURF|2023|Genetically-Programmed Synthetic Cells and Multi-Cellular Machines}}
| [[NSF Cell Free]]
| TBD
| Multiple projects may be available; competitive selection
|}

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-16T01:05:38Z

Mengcao: /* Aim 1: Cloning of modular parts. */

'''[[SURF 2024|SURF 2024]] project description'''
[[File:.jpg|thumb|500px|right|Figure 1: ]]
* Mentor: Mengyi Cao

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with species-specific mutualistic symbiotic bacteria in ''Xenorhabdus'' spp and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they are discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no transgenesis protocols in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be powerful in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit construction (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in S. hermaphroditum, a recently isolated nematode species with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon piggyBac, which will be used to insert the constructs into the targeted sites in ''S. hermaphroditum'' genome. The modular part library will be stored in E. coli and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).

SURF 2024: Establish synthetic biology toolkits for Steinernema nematode transgene expression

2023-12-16T01:05:04Z

Mengcao: Created page with "'''SURF 2024 project description''' Figure 1: * Mentor: Mengyi Cao == '''Project Overview:''' == ''Steinernema'' nematodes associate with species-specific mutualistic symbiotic bacteria in ''Xenorhabdus'' spp and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productiv..."

'''[[SURF 2024|SURF 2024]] project description'''
[[File:.jpg|thumb|500px|right|Figure 1: ]]
* Mentor: Mengyi Cao

== '''Project Overview:''' ==

''Steinernema'' nematodes associate with species-specific mutualistic symbiotic bacteria in ''Xenorhabdus'' spp and together they can infect insects that are agricultural pests. Therefore, the nematode-bacteria partnership is a valuable experimental system to study symbiosis and is important in promoting agricultural productivity. Although they are discovered a century ago, the molecular genetic tools in ''Steinernema'' nematodes are still limiting. Currently there is no transgenesis protocols in these organisms, which severely restricted the visualization and control of gene expression.

Here are a few rate-limiting steps in developing such technique:
# Seek for suitable promoters that are native to the species.
# Optimize the combination of regulatory elements.
# Choose the efficient selection/screen markers.
# Reduce the labor for transgene delivery.

Recently, synthetic biology toolkits, such as a part library, is proven to be powerful in designing various combinations of bacterial genetic elements which rapidly optimized the multigene biocircuit construction (1–3). If similar synthetic biology pipeline can be adapted to multi-cellular eukaryotic organisms, such as nematodes, it will speed up transgenesis tool development and enable the study of ubiquitous and tissue-specific gene expression. In this project, we propose to establish a part library in S. hermaphroditum, a recently isolated nematode species with a complete genome and is available for basic CRISPR-Cas9 genome editing (4,5).

== '''Specific Aims:''' ==

=== Aim 1: Cloning of modular parts.===
We will construct a small-scale library by first cloning modular parts, including (1a): regulatory elements such as various lengths of promoters, 3’UTR sequences, and the first intron; (1b): screen or selection markers, such as codon-optimized fluorescence proteins and hygromycin-resistance cassette; and (1c): ‘inserting sequences’ including homology arms for CRISPR-Cas9, or ITR for transposon piggyBac, which will be used to insert the constructs into the targeted sites in S. hermaphroditum genome. The modular part library will be stored in E. coli and organized in 96-wells plates.

=== Aim 2: Transgene part library construction.===
We will use a Golden Gate-Gibson (3G) cloning pipeline to construct combinations of the modular parts (from Aim 1) and build a library.

=== Aim 3: Transgene delivery ''in vivo''.===
We will deliver these constructs via gonadal microinjection, either based on CRISPR-Cas9, extrachromosomal array, or transposition. We will also attempt a pilot experiment using a liposome-based transfection and electroporation. The latter two delivery approaches, if successful, will significantly relieve the labor from microinjection, and speed up the molecular genetics tool development in ''Steinernema'' and potentially other nematode species.

This proposed effort will be the first step to build comprehensive bioengineering toolkits that will be crucial to tissue-specific gene expression visualization, quantification, and manipulation. If successful at a small scale, the transgene part library can be expanded to a larger scale to build other tools, such as tissue-specific CRISPR, Gal4-UAS gene manipulation, and optogenetics tools.

== '''Preferred Candidate: (fulfills one or more of the following)'''==

* Previous experience with molecular cloning
* Bioengineering-oriented
* Interested in nematode-bacteria symbiosis system
* Basic knowledge in genetics

=='''References:'''==
# Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765.
# Iverson SV, Haddock TL, Beal J, Densmore DM. CIDAR MoClo: Improved MoClo Assembly Standard and New E. coli Part Library Enable Rapid Combinatorial Design for Synthetic and Traditional Biology. ACS Synth Biol. 2016;5(1):99–103.
# Halleran AD, Swaminathan A, Murray RM. Single Day Construction of Multigene Circuits with 3G Assembly. Acs Synth Biol. 2018;7(5):1477–80.
# Cao M. CRISPR-Cas9 genome editing in ''Steinernema'' entomopathogenic nematodes. bioRxiv. 2023;2023.11.24.568619.
# 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. 2021;220(1).