Team:Lethbridge/Part Collection

The Next vivo system is made up of >50 unique parts encoding both protein and RNA components. The main focus of our team this year was to submit all 38 TX-TL protein components to the registry as a collection of parts that anyone could use to produce their own Next vivo system.

Several of these TX-TL components were already in the registry but were under different expression control, lacked purification tags, and/or were not codon optimized for expression in E. coli. As such we sought to improve these parts for future use.

To see our collection on the Standard Registry of Parts, click here.


What is in the Part Collection?

Our part collection will contain all the major biomolecules required for a cell-free transcription-translation system providing them in an easy to express and purify way that anyone can easily produce for their experimental purposes. These biomolecules include:

  • 38 Transcription and Translation (TX-TL) protein genes codon optimized for E. coli expression
  • 20 Next vivo MS2-tagged tRNA isolation genes
  • 2 MS2-tagged ribosomal rRNA genes for isolation of functional ribosomes from the cell lysate

TX-TL Proteins: The initial focus for this collection was submitting all 38 TX-TL protein genes, codon optimized, hexahistidine tagged, and under the control of a T7 promoter system for controlled expression. We have submitted and characterized 9 of these parts to the registry in pSB1C3, characterized expression of 17 parts, and are working toward completing the remaining parts following the competition.

tRNA: The tRNA component of the Next vivo system was another of our focuses for this years project. Currently, no simple protocol exists for efficient and specific isolation of tRNA from the cell. We are happy to announce that we were able to develop a system that allows for the purification of specific, fully modified tRNA from cell lysate. The first tRNA in our part collection is that of tRNAPhe. With the success of our purification strategy, we plan to synthesize the remaining tRNA in our expression construct for submission into this collection.

Ribosomes: Currently we plan to BioBrick both the 23S and 16S E. coli ribosomal rRNA genes with the MS2 hairpin added into either sequence at a region that places it externally on the 3D ribosome structure. This design has previously been used before by another research lab to purify whole ribosomes using the MS2 binding protein tagged with a hexahistidine tag.

MS2BP: The MS2 binding protein is a central component to our tRNA and ribosome purification strategies. It was previously submitted by the 2016 Lethbridge iGEM team and we feel it would be a great addition to our Next vivo Part Collection.

Validation: Finally, we have also included our measurement control construct for the complete TX-TL system. This part expresses a RNA Spinach tagged EYFP mRNA and EYFP protein, that in the addition of DFHBI produces both green and yellow fluorescence indicating transcription and translation respectively.

Part Design Overview

Each TX-TL gene in our collection was designed with several features in mind:

  • Optimized for expression in E. coli
  • N- or C-terminal hexahistidine affinity tag
  • Constructs contain the T7 promoter (BBa_I719005), medium RBS (BBa_B0034), and double terminator (BBa_B0015)

The tRNA genes in our collection were specially designed with several features in mind to facilitate purification:

  • Two MS2 Hairpins transcribed downstream of the tRNA
  • A spacer sequence between the scar sequence and MS2 hairpins where a DNA oligo hybridizes to allow for specific cleavage releasing the tRNA after purification
  • Constructs contain the T7 promoter (BBa_J64997) and a double terminator (BBa_B0015)

Future Use for iGEM

Our hopes are that going forward, teams will be able to use our Next vivo system in the design of their projects. Whether they require a cell-free system to run constructs or just want to test constructs outside of their host organism, Next vivo can be that tool! Designing the system to be purified easily and open-source was key design feature to make this technology available to anyone who wants a cell-free system.

We envision Next vivo continually improving through the addition of new modules and features that can expand the functionalities of the system. These range from accepting non-standard amino acids, to thermostable homologs of each TX-TL protein, to proteins that aid in folding of difficult proteins.

Our Parts in the Registry

BioBrick Protein Full Construct
BBa_K2443000 AlaRS pT7-mRBS-AlaRS-dT
BBa_K2443001 ArgRS pT7-mRBS-ArgRS-dT
BBa_K2443002 AsnRS pT7-mRBS-AsnRS-dT
BBa_K2443003 AspRS pT7-mRBS-AspRS-dT
BBa_K2443004 CysRS pT7-mRBS-CysRS-dT
BBa_K2443005 GlnRS pT7-mRBS-GlnRS-dT
BBa_K2443006 GluRS pT7-mRBS-GluRS-dT
BBa_K2443007 GlyRS alpha subunit pT7-mRBS-GlyRSAlpha-dT
BBa_K2443008 GlyRS beta subunit pT7-mRBS-GlyRSBeta-dT
BBa_K2443009 HisRS pT7-mRBS-HisRS-dT
BBa_K2443010 IleRS pT7-mRBS-IleRS-dT
BBa_K2443011 LeuRS pT7-mRBS-LeuRS-dT
BBa_K2443012 LysRS pT7-mRBS-LysRS-dT
BBa_K2443013 MetRS pT7-mRBS-MetRS-dT
BBa_K2443014 PheRS alpha subunit pT7-mRBS-PheRSAlpha-dT
BBa_K2443015 PheRS beta subunit pT7-mRBS-PheRSBeta-dT
BBa_K2443016 ProRS pT7-mRBS-ProRS-dT
BBa_K2443017 SerRS pT7-mRBS-SerRS-dT
BBa_K2443018 ThrRS pT7-mRBS-ThrRS-dT
BBa_K2443019 TrpRS pT7-mRBS-TrpRS-dT
BBa_K2443020 TyrRS pT7-mRBS-TyrRS-dT
BBa_K2443021 ValRS pT7-mRBS-ValRS-dT
BBa_K2443022 MTF pT7-mRBS-MTF-dT
BBa_K2443023 IF1 pT7-mRBS-IF1-dT
BBa_K2443024 IF2 pT7-mRBS-IF2-dT
BBa_K2443025 IF3 pT7-mRBS-IF3-dT
BBa_K2443026 EF-G pT7-mRBS-EFG-dT
BBa_K2443027 EF-Tu pT7-mRBS-EFTu-dT
BBa_K2443028 EF-Ts pT7-mRBS-EFTs-dT
BBa_K2443029 RF1 pT7-mRBS-RF1-dT
BBa_K2443030 RF2 pT7-mRBS-RF2-dT
BBa_K2443031 RF3 pT7-mRBS-RF3-dT
BBa_K2443032 RRF pT7-mRBS-RRF-dT
BBa_K2443033 MK pT7-mRBS-MK-dT
BBa_K2443034 CK pT7-mRBS-CK-dT
BBa_K2443035 NDK pT7-mRBS-NDK-dT
BBa_K2443036 PPiase pT7-mRBS-PPiase-dT
BBa_K2443037 T7 RNA Polymerase pT7-mRBS-T7 RNA Polymerase-dT
BBa_K2443038 tRNAPhe pT7-tRNAPhe-2xMS2 Hairpin-dT
BBa_K2443039 EYFP-Spinach TX-TL Measurement Construct pT7-mRBS-EYFP-Spinach-dT
BBa_K2109108 MS2BP pT7-mRBS-MS2BP-dT

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