Team:Lethbridge/Design
Design Objectives
Our design was guided by the needs of three user groups:
Simple scientific protocols
Standardized parts
Well characterized and developed system
Standard, easy to use system
Simplified protocols
Modular tool that can be tailored to the application
Modular experiments
Open-source
Open-source
Promote good safety practices
Standardized parts
Resulting in the identification of four core design considerations for our system
STANDARD, MODULAR, SAFE, and USER-FRIENLDY
Overview
To ensure utility of our system, we developed a simplified expression and purification strategy to produce the necessary biomachinery for cell-free synthetic biology.
Our system includes all of the 36 essential TX-TL proteins, the 23S and 16S rRNA, and a tRNA for each amino acid, where tRNAPhe will act as a proof of concept for our novel purification method. In addition, we include the MS2 coat protein which is essential to our tRNA and rRNA purification strategies.
In total we have designed 41 parts! (link to parts pages)
Standard Construct Design
Each construct consisted of a T7 promoter (BBa_I719005), medium ribosome binding site (RBS) (BBa_B0034), protein coding sequence, serine-glycine linker and histidine tag at the N- or C-terminus, and a double terminator (BBa_B0015).
The promoter, RBS and terminator are standard BioBrick parts well-characterized in the registry, providing a good starting point for the initial characterization of our system. Each construct was also codon optimized to allow for efficient expression in E. coli.
Purification Strategy
Protein Purification
Transcription and translation factors are expressed with a 6 histidine residue tag to allow for simple purification by nickel sepharose affinity chromatography. The polyhistidine tag is encoded at the N- or C-terminus of each gene based on what was previously described in the literature (ref).
RNA Purification
To purify RNA, we take advantage of a previously published purification strategy adapted from the MS2 bacteriophage (Ref). It takes advantage of a specific RNA:protein interaction between an RNA hairpin in the MS2 genome and the MS2 coat protein (MS2BP) that forms the viral capsid. The MS2BP has a polyhistidine tag expressed at its C-terminus. This alteration gives the MS2 coat protein dual binding ability. It will have high affinity to nickel as well as to the MS2 hairpin. Thus, tRNA and rRNA expressing an MS2 hairpin can be purified in the same column as the polyhistidine tagged proteins.
We developed a novel purification system based on a general construct, whereby an RNA of interest will be transcribed with a scar and spacer region, acting as a oligo binding site. A DNA oligo can bind to this site, resulting in a DNA-RNA hybrid, as a target for cleavage by RNase H. This will be followed by two MS2 hairpins, enabling tight binding to the MS2BP for purification.
Our system is designed such that all components can be purified at the same time by nickel sepharose affinity chromatography!
tRNA
Ribosomes
Validation Construct
Next vivo 2.0
