Team:Lethbridge/Demonstrate

Next vivo was designed in such a way that we could test multiple components and modules individually while developing the full system. These include:

• Evaluating the efficacy of multiprotein purification using TX-TL proteins
• Developing a universal tool to characterize TX-TL systems
• Designing and testing a novel tRNA purification strategy
• Testing the TX components of our system for activity

Multi-protein Purification

One of the key features of Next vivo is the ability purify all the components in a single step purification. As a proof of concept we expressed four of the TX-TL components and co-purified them all using a nickel sepharose chromatography column (see figure below). The four proteins used in this initial test were selected based on their molecular weights relative to each other for visualization purposes.

Josh’s excellent gel of both overexpression and purification awesomeness.

Figure 1 - Representative overexpression and multiprotein purification of TX-TL components. Each TX-TL component was expressed from E. coli cells carrying the plasmid encoding the specified component and samples three hours post induction were collected (Lane 2-5). The expressing cells of each component were pooled and lysed before applying the lysate to a nickel-sepharose affinity column for isolation of just the polyhistidine tagged TX-TL components. After washing away the unwanted cellular proteins and debris, the TX-TL components were eluted from the Nickel-sepharose to a high level of purity (Lane 6).

From this initial test we have confidence that scaling the multi-protein purification up to include all, or large groups of, the TX-TL proteins will be feasible. The overexpression and purification of these four proteins was done with minimal lab equipment and supplies.

Validation Construct

tRNA Purification

Transcription Validation

Future Plans