Murray Wiki - User contributions [en]

RMM research meetings, May 2025

2025-05-08T21:19:34Z

Yz473: change Yan's meeting time

Please sign up for a slot below.

5 May (Mon):
* 8:30 am: Ioannis
* 9:15 am: Leo
* 3 pm: Josefine
* 3:45 pm: Han

12 May (Mon):
* 10 am: Nikos
* 10:45 am: Zach M
* 12:30 pm: Alex J.
* 3:30 pm: David
* 4:15 pm: open
* 5:00 pm: Yan

RMM research meetings, May 2025

2025-04-28T19:28:19Z

Yz473:

Please sign up for a slot below.

5 May (Mon):
* 8:30 am: Ioannis
* 9:15 am: Leo
* 3 pm: Josefine
* 3:45 pm: Han

11 May (Sun):
* 2:15 pm: Open
* 3 pm: Open
* 3:45 pm: Open
* 4:30 pm: Open

12 May (Mon):
* 10 am: Nikos
* 10:45 am: Zach M
* 3:30 pm: Open
* 4:15 pm: Yan

SURF discussions, Jan 2024

2024-01-21T21:33:19Z

Yz473: /* 22 Jan (Mon) */

Slots for talking with applicants and co-mentors about SURF projects. Please sign up for one of the slots below. All times are PST. __NOTOC__

In preparation for our conversation, please do the following:
* SURF students should work with their co-mentors to find a time the meeting/Zoom call. (For Zoom calls, co-mentors should initiate.)
* Please make sure you have read the material in the description of your project, so that you are prepared to talk about what the project is about and we can narrow in on the key ideas that will be the basis of your proposal
* Please take a look at the [[SURF GOTChA chart]] page, which is the format that we will use for the first iteration of your project proposal. It would be great to show up with a first draft of your GOTChA chart.

{| border=1 width=100%
|- valign=top
| width=33% |
==== 22 Jan (Mon) ====
* 10:15-10:45 am PST: open
* 12:30-1:00 pm PST: open
* 3:00-3:30 pm PST: open
* 3:30-4:00 pm PST: Lovisa, Yan, Zach

| width=33% |

==== 23 Jan (Tue) ====
* 2:00-2:30 pm PST: open
* 2:30-3:00 pm PST: open
* 3:00-3:30 pm PST: open

| width=33% |

==== 24 Jan (Wed) ====
* 9:15-9:45 am PST: open
* 12:45-1:15 pm PST: open

|}

The agenda for the phone call is (roughly):

# Description of the basic idea behind the project (based on applicant's understanding)
# Discussion about approaches, what you have read, variations to consider, etc
# Review of GOTChA chart and how we will use it
# Discussion of the format of the proposal
# Questions and discussion about the process

SURF 2024: Making Purer PURE

2023-12-30T02:01:22Z

Yz473: /* Project Overview */

'''[[SURF 2024|2024 SURF]] project description'''
__NOTOC__
* Mentor: Richard Murray
* Co-mentors: Yan Zhang and Zachary Martinez

Main Objective: Reduce background contaminant of One-Pot PURE systems

=== Introduction ===
The PURE (Protein Synthesis Using Recombinant Enzymes) system uses a defined set of 36 enzymes necessary for transcription, translation, and energy cycling to create a minimal system to support protein expression. This minimal system allows facile removal or replacements of specific enzymes involved in reactions with the potential to power impactful applications in prototyping novel protein designs via complementation assays and producing therapeutic proteins via codon replacement for non-canonical amino acids.

Despite the promise of PURE, current homemade One-Pot PURE systems relying on metal-affinity-based purification exhibit high background containments in the purified product. Although traditional single protein purification methods can be interfaced with downstream chromatography steps to achieve high product purity, such processing steps are incompatible with the purification of One-Pot PURE, which involves the simultaneous purification of 36 proteins spanning 7 – 110 kDa. To equip researchers with the capability to develop truly defined and pure PURE systems, a new method to create a purer PURE system is necessary.

=== Motivation ===
Current homemade and One-Pot PURE systems rely on traditional 6xHis tags for protein purification. This results in high background contaminants in the purified product – particularly metal-binding proteins. To enable impactful biological applications where the absence of background contaminants is critical, we need an approach to developing the purer PURE system. The central hypothesis of this project is to investigate the use of peptide- and protein-based tags to produce a PURE system without background contamination.

===Project Overview ===
This project investigates whether peptide- and protein-based purification tags can lead to a purer PURE system. The key milestones of the projects are delineated as follows:

1. To prevent the purification of truncated products, this project is limited to identifying suitable affinity tags for C-terminal incorporation.

2. Selecting optimal purification tag candidates that minimize disruption to protein folding and enzymatic activity using predictive protein structure models.

3. Cloning screened purification tag candidates for 36 enzymes constituting the PURE system.

4. Verifying that the One-Pot PURE system made with new affinity tags has significantly less background protein contamination than the traditional his-tagged method using mass spectrometry-based proteomics.

5. Characterizing the protein yield and reaction kinetics of traditional and “purer” One-Pot PURE systems.

=== Preferred Skills ===
<li> Background in basic molecular biology through coursework </li>
<li> Some experimental experience through coursework with a lab component or research </li>
<li> Students with structural biology background are highly preferred </li>

=== References ===
Lavickova, B. and Maerkl, S. J. (2019). A simple, robust, and low-cost method to produce the pure cell-free system. ACS Synthetic Biology, 8(2), 455-462. https://doi.org/10.1021/acssynbio.8b00427

SURF 2024: Making Purer PURE

2023-12-30T02:00:53Z

Yz473:

'''[[SURF 2024|2024 SURF]] project description'''
__NOTOC__
* Mentor: Richard Murray
* Co-mentors: Yan Zhang and Zachary Martinez

Main Objective: Reduce background contaminant of One-Pot PURE systems

=== Introduction ===
The PURE (Protein Synthesis Using Recombinant Enzymes) system uses a defined set of 36 enzymes necessary for transcription, translation, and energy cycling to create a minimal system to support protein expression. This minimal system allows facile removal or replacements of specific enzymes involved in reactions with the potential to power impactful applications in prototyping novel protein designs via complementation assays and producing therapeutic proteins via codon replacement for non-canonical amino acids.

Despite the promise of PURE, current homemade One-Pot PURE systems relying on metal-affinity-based purification exhibit high background containments in the purified product. Although traditional single protein purification methods can be interfaced with downstream chromatography steps to achieve high product purity, such processing steps are incompatible with the purification of One-Pot PURE, which involves the simultaneous purification of 36 proteins spanning 7 – 110 kDa. To equip researchers with the capability to develop truly defined and pure PURE systems, a new method to create a purer PURE system is necessary.

=== Motivation ===
Current homemade and One-Pot PURE systems rely on traditional 6xHis tags for protein purification. This results in high background contaminants in the purified product – particularly metal-binding proteins. To enable impactful biological applications where the absence of background contaminants is critical, we need an approach to developing the purer PURE system. The central hypothesis of this project is to investigate the use of peptide- and protein-based tags to produce a PURE system without background contamination.

===Project Overview ===
This project investigates whether peptide- and protein-based purification tags can lead to a purer PURE system. The key milestones of the projects are delineated as follows:

1. To prevent the purification of truncated products, this project is limited to identifying suitable affinity tags for C-terminal incorporation.
2. Selecting optimal purification tag candidates that minimize disruption to protein folding and enzymatic activity using predictive protein structure models
3. Cloning screened purification tag candidates for 36 enzymes constituting the PURE system.
4. Verify that the One-Pot PURE system made with new affinity tags has significantly less background protein contamination than the traditional his-tagged method using mass spectrometry-based proteomics.
5. Characterize and compare the protein yield and reaction kinetics of traditional and “purer” One-Pot PURE systems.

=== Preferred Skills ===
<li> Background in basic molecular biology through coursework </li>
<li> Some experimental experience through coursework with a lab component or research </li>
<li> Students with structural biology background are highly preferred </li>

=== References ===
Lavickova, B. and Maerkl, S. J. (2019). A simple, robust, and low-cost method to produce the pure cell-free system. ACS Synthetic Biology, 8(2), 455-462. https://doi.org/10.1021/acssynbio.8b00427

SURF 2024: Making Purer PURE

2023-12-30T02:00:32Z

Yz473: /* References */

'''[[SURF 2024|2024 SURF]] project description'''
__NOTOC__
* Mentor: Richard Murray
* Co-mentors: Yan Zhang and Zachary Martinez

Main Objective: Reduce background contaminant of One-Pot PURE systems

=== Introduction ===
The PURE (Protein Synthesis Using Recombinant Enzymes) system uses a defined set of 36 enzymes necessary for transcription, translation, and energy cycling to create a minimal system to support protein expression. This minimal system allows facile removal or replacements of specific enzymes involved in reactions with the potential to power impactful applications in prototyping novel protein designs via complementation assays and producing therapeutic proteins via codon replacement for non-canonical amino acids.

Despite the promise of PURE, current homemade One-Pot PURE systems relying on metal-affinity-based purification exhibit high background containments in the purified product. Although traditional single protein purification methods can be interfaced with downstream chromatography steps to achieve high product purity, such processing steps are incompatible with the purification of One-Pot PURE, which involves the simultaneous purification of 36 proteins spanning 7 – 110 kDa. To equip researchers with the capability to develop truly defined and pure PURE systems, a new method to create a purer PURE system is necessary.

=== Motivation ===
Current homemade and One-Pot PURE systems rely on traditional 6xHis tags for protein purification. This results in high background contaminants in the purified product – particularly metal-binding proteins. To enable impactful biological applications where the absence of background contaminants is critical, we need an approach to developing the purer PURE system. The central hypothesis of this project is to investigate the use of peptide- and protein-based tags to produce a PURE system without background contamination.

===Project Overview ===
This project investigates whether peptide- and protein-based purification tags can lead to a purer PURE system. The key milestones of the projects are delineated as follows:

1. To prevent the purification of truncated products, this project is limited to identifying suitable affinity tags for C-terminal incorporation.
2. Selecting optimal purification tag candidates that minimize disruption to protein folding and enzymatic activity using predictive protein structure models
3. Cloning screened purification tag candidates for 36 enzymes constituting the PURE system.
4. Verify that the One-Pot PURE system made with new affinity tags has significantly less background protein contamination than the traditional his-tagged method using mass spectrometry-based proteomics.
5. Characterize and compare the protein yield and reaction kinetics of traditional and “purer” One-Pot PURE systems.

=== Preferred Skills ===
<ul>
<li> Background in basic molecular biology through coursework </li>
<li> Some experimental experience through coursework with a lab component or research </li>
<li> Students with structural biology background are highly preferred </li>

=== References ===
Lavickova, B. and Maerkl, S. J. (2019). A simple, robust, and low-cost method to produce the pure cell-free system. ACS Synthetic Biology, 8(2), 455-462. https://doi.org/10.1021/acssynbio.8b00427

SURF 2024: Making Purer PURE

2023-12-30T02:00:15Z

Yz473: Created page with "'''2024 SURF project description''' __NOTOC__ * Mentor: Richard Murray * Co-mentors: Yan Zhang and Zachary Martinez Main Objective: Reduce background contaminant of One-Pot PURE systems === Introduction === The PURE (Protein Synthesis Using Recombinant Enzymes) system uses a defined set of 36 enzymes necessary for transcription, translation, and energy cycling to create a minimal system to support protein expression. This minimal system allows fa..."

'''[[SURF 2024|2024 SURF]] project description'''
__NOTOC__
* Mentor: Richard Murray
* Co-mentors: Yan Zhang and Zachary Martinez

Main Objective: Reduce background contaminant of One-Pot PURE systems

=== Introduction ===
The PURE (Protein Synthesis Using Recombinant Enzymes) system uses a defined set of 36 enzymes necessary for transcription, translation, and energy cycling to create a minimal system to support protein expression. This minimal system allows facile removal or replacements of specific enzymes involved in reactions with the potential to power impactful applications in prototyping novel protein designs via complementation assays and producing therapeutic proteins via codon replacement for non-canonical amino acids.

Despite the promise of PURE, current homemade One-Pot PURE systems relying on metal-affinity-based purification exhibit high background containments in the purified product. Although traditional single protein purification methods can be interfaced with downstream chromatography steps to achieve high product purity, such processing steps are incompatible with the purification of One-Pot PURE, which involves the simultaneous purification of 36 proteins spanning 7 – 110 kDa. To equip researchers with the capability to develop truly defined and pure PURE systems, a new method to create a purer PURE system is necessary.

=== Motivation ===
Current homemade and One-Pot PURE systems rely on traditional 6xHis tags for protein purification. This results in high background contaminants in the purified product – particularly metal-binding proteins. To enable impactful biological applications where the absence of background contaminants is critical, we need an approach to developing the purer PURE system. The central hypothesis of this project is to investigate the use of peptide- and protein-based tags to produce a PURE system without background contamination.

===Project Overview ===
This project investigates whether peptide- and protein-based purification tags can lead to a purer PURE system. The key milestones of the projects are delineated as follows:

1. To prevent the purification of truncated products, this project is limited to identifying suitable affinity tags for C-terminal incorporation.
2. Selecting optimal purification tag candidates that minimize disruption to protein folding and enzymatic activity using predictive protein structure models
3. Cloning screened purification tag candidates for 36 enzymes constituting the PURE system.
4. Verify that the One-Pot PURE system made with new affinity tags has significantly less background protein contamination than the traditional his-tagged method using mass spectrometry-based proteomics.
5. Characterize and compare the protein yield and reaction kinetics of traditional and “purer” One-Pot PURE systems.

=== Preferred Skills ===
<ul>
<li> Background in basic molecular biology through coursework </li>
<li> Some experimental experience through coursework with a lab component or research </li>
<li> Students with structural biology background are highly preferred </li>

===References ===
Lavickova, B. and Maerkl, S. J. (2019). A simple, robust, and low-cost method to produce the pure cell-free system. ACS Synthetic Biology, 8(2), 455-462. https://doi.org/10.1021/acssynbio.8b00427

SURF 2024

2023-12-30T01:56:32Z

Yz473: /* 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|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|2024|Towards a Minimal Model for Virus-Host Interactions}}
| TBD
| Zachary Martinez
|
|-
|| {{SURF|2024|Making Purer PURE}}
| TBD
| Yan Zhang and Zachary Martinez
|-
| {{SURF|2023|Genetically-Programmed Synthetic Cells and Multi-Cellular Machines}}
| [[NSF Cell Free]]
| TBD
| Multiple projects may be available; competitive selection
|}

Andras Gyorgy, Aug 2023

2023-08-07T20:48:53Z

Yz473: /* Schedule */

Andras Gyorgy from NYU Abu Dhabi will visit on 7 Aug (Mon).

== Schedule ==

* 9:30 am: Richard, 109 Steele
* 10:00 am: Manisha, 138 Keck
* 10:30 am: Zach Martinez, 138 Keck
* 11:00 am: Seminar, 111 Keck
* 12:15 pm: Lunch with graduate students (organized by John M)
* 1:30 pm: Matt K
* 2:15 pm: Yan, 138 Keck
* 3:00 pm: John M, location TBD
* 3:45 pm: Inigo, Annenberg 2nd floor lounge
* 4:30 pm: Richard, 109 Steele

Andras Gyorgy, Aug 2023

2023-08-03T15:58:28Z

Yz473: /* Schedule */

Andras Gyorgy from NYU Abu Dhabi will visit on 7 Aug (Mon).

== Schedule ==

* 9:30 am: Richard, 109 Steele
* 10:00 am: John Marken, location TBD
* 10:30 am: Zach Martinez, Red Door(?)
* 11:00 am: Seminar, 111 Keck
* 12:15 pm: Lunch with graduate students
* 1:30 pm: open
* 2:15 pm: Yan, location TBD
* 3:00 pm: open
* 3:45 pm: Inigo, Annenberg 2nd floor lounge
* 4:30 pm: Richard, 109 Steele

SURF discussions, Feb 2023

2023-01-24T22:49:23Z

Yz473: /* 7 Feb (Tue) */

Slots for talking with applicants and co-mentors about SURF projects. Please sign up for one of the slots below. All times are PST. __NOTOC__

In preparation for our conversation, please do the following:
* SURF students should work with their co-mentors to find a time the meeting/Zoom call. (For Zoom calls, co-mentors should initiate.)
* Please make sure you have read the material in the description of your project, so that you are prepared to talk about what the project is about and we can narrow in on the key ideas that will be the basis of your proposal
* Please take a look at the [[SURF GOTChA chart]] page, which is the format that we will use for the first iteration of your project proposal. It would be great to show up with a first draft of your GOTChA chart.

{| border=1 width=100%
|- valign=top
| width=50% |
==== 6 Feb (Mon) ====
* 8:00 am PST: open
* 8:30 am PST: open
<hr>
* 1:00 pm PST: open
* 1:30 pm PST: Phillipe and Zach
| width=50% |

==== 7 Feb (Tue) ====
* 8:00 am PST: open
* 8:30 am PST: open
<hr>
* 5:00 pm PST: Kelly & Yan
* 5:30 pm PST: open

|}

The agenda for the phone call is (roughly):

# Description of the basic idea behind the project (based on applicant's understanding)
# Discussion about approaches, what you have read, variations to consider, etc
# Review of GOTChA chart and how we will use it
# Discussion of the format of the proposal
# Questions and discussion about the process

SURF 2023: Lysate Optimization to Extend Cell-Free Reaction Lifetime

2022-12-20T19:45:54Z

Yz473:

'''[[SURF 2023|2023 SURF]] project description'''

*Mentor: Richard Murray
*Co-mentor: Yan Zhang

=== Introduction: ===

Cell-free expression systems harness the transcriptional and translational machinery of living cells to enable in vitro functions that are usually only effective in vivo [1]. Cell-free reactions can be created from purified recombinant elements comprising the minimum set of necessary transcriptional and translational machinery (PURE systems) [2] or via crude cell lysate from cells [3]. Of which, the crude cell lysate-based system, particularly those harvested from E. coli cells, is more widely used and cost-effective.

However, a significant gap exists in understanding the background metabolism in lysate-based cell-free systems. Recent works have shown that endogenous metabolism is active cell-free lysates, regardless of whether the reaction produces proteins [4, 5]. Thus, being able to identify major “waste” pathways and reroute energy consumption to focus only on protein production can potentially extend the lifetime of cell-free reactions.

=== Project Overview: ===

E. coli BL21 will be the primary strain for lysate-based cell-free protein expression. The SURF student will first identify potential gene candidates for “waste” metabolic branches responsible for diverting energy away from protein synthesis. Following branch point identification, the student will determine the optimal method for “waste” pathway removal. For example, the relevant enzyme involved in a pathway’s entry point can be removed via gene knockout [6]. However, these gene knockouts can also make cells non-viable for culturing during the lysate preparation. To circumvent this problem, alternative strategies, such as inserting affinity tags and removing proteins via affinity columns, will be explored [7]. Once gene candidates and their optimal method of removal are determined, the student will be trained by the co-mentor to carry out gene removal, cell-free lysate preparation, cell-free reaction assembly, and metabolite profile assessments.

=== SURF Student Qualifications: ===

Prerequisite coursework: Bi1x (or a similar introductory biology course that provides a basic foundation in molecular biology and biochemistry)

Preferred coursework: BE 150, BE/APh 161, and at least one biology course with a lab component

Other: basic familiarity with coding preferred to aid in experiment design and data analysis (Python, etc.)

=== References: ===

[1] Gregorio, N.E.; Levine, M.Z.; Oza, J.P. A User’s Guide to Cell-Free Protein Synthesis. Methods Protoc. 2019, 2, 24. https://doi.org/10.3390/mps2010024

[2] Lavickova, B. and Maerkl, S.J. A Simple, Robust, and Low-Cost Method to Produce the PURE Cell-Free System. ACS Synthetic Biology 2019 8 (2), 455-462. https://doi.org/10.1021/acssynbio.8b00427

[3] Garenne, D., Haines, M.C., Romantseva, E.F. et al. Cell-free gene expression. Nat Rev Methods Primers 1, 49 (2021). https://doi.org/10.1038/s43586-021-00046-x

[4] Miguez, A.M., McNerney, M.P., Styczynski, M.P., Metabolic Profiling of Escherichia coli-Based Cell-Free Expression Systems for Process Optimization. Industrial & Engineering Chemistry Research 2019 58 (50), 22472-22482. https://doi.org/10.1021/acs.iecr.9b03565

[5] Miguez, A.M., Zhang, Y., Piorino, F., Styczynski, M.P., Metabolic Dynamics in Escherichia coli-Based Cell-Free Systems. ACS Synthetic Biology 2021 10 (9), 2252-2265. https://doi.org/10.1021/acssynbio.1c00167

[6] Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5. https://doi.org/10.1073/pnas.120163297

[7] Garcia, D.C., Dinglasan, J.L.N, Shrestha, H., Abraham, P.E., Hettich, R.L, Doktycz, M.J., Lysate proteome engineering strategy for enhancing cell-free metabolite production, Metabolic Engineering Communications, 12, 2021, e00162,
https://doi.org/10.1016/j.mec.2021.e00162

SURF 2023: Lysate Optimization to Extend Cell-Free Reaction Lifetime

2022-12-20T07:27:50Z

Yz473: Created page with "Mentor: Richard Murray Co-mentor: Yan Zhang Introduction: Cell-free expression systems harness the transcriptional and translational machinery of living cells to enable in vitro functions that are usually only effective in vivo [1]. Cell-free reactions can be created from purified recombinant elements comprising the minimum set of necessary transcriptional and translational machinery (PURE systems) [2] or via crude cell lysate from cells [3]. Of which, the..."

Mentor: Richard Murray

Co-mentor: Yan Zhang

 Introduction: 

Cell-free expression systems harness the transcriptional and translational machinery of living cells to enable in vitro functions that are usually only effective in vivo [1]. Cell-free reactions can be created from purified recombinant elements comprising the minimum set of necessary transcriptional and translational machinery (PURE systems) [2] or via crude cell lysate from cells [3]. Of which, the crude cell lysate-based system, particularly those harvested from E. coli cells, is more widely used and cost-effective.

However, a significant gap exists in understanding the background metabolism in lysate-based cell-free systems. Recent works have shown that endogenous metabolism is active cell-free lysates, regardless of whether the reaction produces proteins [4, 5]. Thus, being able to identify major “waste” pathways and reroute energy consumption to focus only on protein production can potentially extend the lifetime of cell-free reactions.

 Project Overview: 

E. coli BL21 will be the primary strain for lysate-based cell-free protein expression. The SURF student will first identify potential gene candidates for “waste” metabolic branches responsible for diverting energy away from protein synthesis. Following branch point identification, the student will determine the optimal method for “waste” pathway removal. For example, the relevant enzyme involved in a pathway’s entry point can be removed via gene knockout [6]. However, these gene knockouts can also make cells non-viable for culturing during the lysate preparation. To circumvent this problem, alternative strategies, such as inserting affinity tags and removing proteins via affinity columns, will be explored [7]. Once gene candidates and their optimal method of removal are determined, the student will be trained by the co-mentor to carry out gene removal, cell-free lysate preparation, cell-free reaction assembly, and metabolite profile assessments.

 SURF Student Qualifications: 

Prerequisite coursework: Bi1x (or a similar introductory biology course that provides a basic foundation in molecular biology and biochemistry)

Preferred coursework: BE 150, BE/APh 161, and at least one biology course with a lab component

Other: basic familiarity with coding preferred to aid in experiment design and data analysis (Python, etc.)

 References: 

[1] Gregorio, N.E.; Levine, M.Z.; Oza, J.P. A User’s Guide to Cell-Free Protein Synthesis. Methods Protoc. 2019, 2, 24. https://doi.org/10.3390/mps2010024

[2] Lavickova, B. and Maerkl, S.J. A Simple, Robust, and Low-Cost Method to Produce the PURE Cell-Free System. ACS Synthetic Biology 2019 8 (2), 455-462. https://doi.org/10.1021/acssynbio.8b00427

[3] Garenne, D., Haines, M.C., Romantseva, E.F. et al. Cell-free gene expression. Nat Rev Methods Primers 1, 49 (2021). https://doi.org/10.1038/s43586-021-00046-x

[4] Miguez, A.M., McNerney, M.P., Styczynski, M.P., Metabolic Profiling of Escherichia coli-Based Cell-Free Expression Systems for Process Optimization. Industrial & Engineering Chemistry Research 2019 58 (50), 22472-22482. https://doi.org/10.1021/acs.iecr.9b03565

[5] Miguez, A.M., Zhang, Y., Piorino, F., Styczynski, M.P., Metabolic Dynamics in Escherichia coli-Based Cell-Free Systems. ACS Synthetic Biology 2021 10 (9), 2252-2265. https://doi.org/10.1021/acssynbio.1c00167

[6] Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5. https://doi.org/10.1073/pnas.120163297

[7] Garcia, D.C., Dinglasan, J.L.N, Shrestha, H., Abraham, P.E., Hettich, R.L, Doktycz, M.J., Lysate proteome engineering strategy for enhancing cell-free metabolite production, Metabolic Engineering Communications, 12, 2021, e00162,
https://doi.org/10.1016/j.mec.2021.e00162

Manos Alexis, Oct 2022

2022-10-21T18:45:44Z

Yz473: /* 24 Oct (Mon) */

Manos Alexis, a PhD student at Cambridge interested in dynamical systems, control theory, synthetic biology and systems biology will visit our group the week of 24 Oct. You can sign up for a time to meet with him below:

=== 24 Oct (Mon) ===

* 9:00 am: Monica Nolasco, 107 Steele Lab
* 10:00 am: Richard Murray, 109 Steele Lab
* 10:45 am: Yan Zhang, Red Door Cafe
* 11:30 am: Alex Johnson
* 12:00 pm: Lunch w/ soil syn bio students (tentative)
* 1:30 pm: John Marken
* 2:15 pm: Zoila Jurado, Red Door Cafe
* 3:00 pm: open
* 3:45 pm: open
* 4:30 pm: open

=== 25 Oct (Tue) ===

* 9:00 am: open
* 10:00 am: Biocircuits group meeting, 111 Keck
* 12:00 pm: Lunch w/ synthetic cell students (tentative)
* 1:30 pm: open
* 2:15 pm: open
* 3:00 pm: open
* 3:45 pm: open
* 4:30 pm: open