SURF 2024: Making Purer PURE - Revision history

Yz473: /* Project Overview */

2023-12-30T02:01:22Z

Project Overview

← Older revision		Revision as of 02:01, 30 December 2023
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	1. To prevent the purification of truncated products, this project is limited to identifying suitable affinity tags for C-terminal incorporation.		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
			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.		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.		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 ===		=== Preferred Skills ===

Yz473 at 02:00, 30 December 2023

2023-12-30T02:00:53Z

← Older revision		Revision as of 02:00, 30 December 2023
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	=== Preferred Skills ===		=== Preferred Skills ===
	~~<ul>~~
	<li> Background in basic molecular biology through coursework </li>		<li> Background in basic molecular biology through coursework </li>
	<li> Some experimental experience through coursework with a lab component or research </li>		<li> Some experimental experience through coursework with a lab component or research </li>

Yz473: /* References */

2023-12-30T02:00:32Z

References

← Older revision		Revision as of 02:00, 30 December 2023
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	<li> Students with structural biology background are highly preferred </li>		<li> Students with structural biology background are highly preferred </li>

	===References ===		=== 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		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

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..."

2023-12-30T02:00:15Z

Created page with "'''2024 SURF project description''' __NOTOC__ * Mentor: Richard Murray * Co-mentors: Yan Zhang and Zachary Martinez <b>Main Objective:</b> 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..."

New page

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

<b>Main Objective:</b> 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