The Next vivo system is made up of >40 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.

In addition to the TX-TL proteins, we also sought to BioBrick the ribosomal RNA genes and twenty tRNA genes. As a proof of concept for our novel tRNA purification design we only synthesized one representative tRNA gene before ordering them all. Additionally, the Next vivo system makes use of a previously submitted MS2BP part (BBa_K2109108).

Part Collection

The Next vivo part collection consists of the 38 TX-TL proteins required for RNA transcription and protein synthesis. Each part in the collection has a hexahistidine tag for simultaneous purification of all components, the T7 promoter (BBa_I719005), medium RBS (BBa_B0034), and double terminator (BBa_B0015).

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

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Parts Submitted

This season we were able to submit 9 parts in pSB1C3 to the registry from our part collection. We aim to continually submit parts as we successfully ligate them into pSB1C3. These include:

Parts in pSB1C3 Table

BioBrick	Protein
BBa_K2443001	ArgRS
BBa_K2443003	AsnRS
BBa_K2443005	GlnRS
BBa_K2443008	GlyRS Beta
BBa_K2443013	MetRS
BBa_K2443024	IF2
BBa_K2443026	EF-G
BBa_K2443031	RF3
BBa_K2443034	CK

Composite Parts

General Protein Construct

General RNA Construct

This year we designed 40 composite parts. Of the 40 parts, there are 38 proteins essential for prokaryotic transcription and translation (TX-TL) as well 2 additional constructs for tRNA purification and TX-TL validation.

Improved Parts

Of our 38 TX-TL components, five were already in the registry of standard biological parts. We decided to improve these parts for future use in our Next vivo system and for general use by others. Our improvements are documented here.

Basic Parts

All basic parts pages created on the registry of standard biological parts. Contains all 38 proteins essential for prokaryotic transcription and translation (TX-TL) as well as the parts for our tRNA purification and TX-TL validation construct.

Previous Parts Used

During the design of our project we utilized several parts already in the registry in our composite parts including:

Existing Parts Used Table

BioBrick	Part
BBa_I719005	T7 promoter
BBa_B0034	medium ribosome binding site (mRBS)
BBa_B0015	Double Terminator
BBa_J64997	T7 -10 and rest
BBa_K1983010	tRNAPhe