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Revision as of 22:07, 17 September 2015

Synbiota

Introduction

We were awarded trailblazer scholarship by Synbiota, a startup company co-founded by iGEM alumni, and had a chance to test their prototype Rapid DNA Prototyping (RDP) assembly standard including exclusive iGEM kit and RDP part designing software.

RDP is a modular approach to synthetic biology that is biobrick compatible, and somewhat similar to the standard biobrick approach in a sense that DNA templates can create new RDP parts through PCR. However, each RDP part is defined by each promoter, RBS, coding region, or terminator. Series of RDP parts can be assembled using the magnetic anchor under two hours.

Synbiota image

Figure 1. Addition of sticky ends at the each strand allows two different RDP parts to be attached.

Design

RDP Standard requires addition of two unique sticky ends (X and Z) and their complements (X’ and Z’) onto each part, allowing sequential assembly from promoter to terminator. Figure 1 shows this process.

We have designed two customised RDP parts, pLsr and T4Holin. These RDP parts were designed using GEntle software to contain X-Z’ or Z-X’ sticky ends. Synbiota has provided us with physical RDP parts of pLse and T4Holin, as well as primers to amplify them through PCR. Wetlab notebooks of Synbiota specific PCR can be found here.

Then pLsr and T4Holin were combined with the provided Anchor RDP part, RBS, terminator and Cap to make a full RDP circuit which is equivalent to BBa_K1659601.

So this is how our part BBa_K1659601 RDP circuit looks like:

Our BBa_K1659601 RDP circuit

Our BBa_K1659601 RDP Circuit

Each ends of Anchor and Cap RDP parts self-assemble to form pSB1C3 backbone that is biobrick compatible.

Assembly

RDP assembly method is very time-efficient because the assembly starts with a Synbiota Anchor part (DNA anchored to a magnetic bead), and first RDP part (pLsr in our case) is added onto the solution, requiring ligation incubation time of only 8min. Then, an eppendorf tube holder with the magnet pellets Anchor-pLsr component, washing off excess DNA and ligase. The picture below illustrates this very stage.

Us assembling our construct using the magnet

Then, next RDP part(RBS 2.1 in this case) resuspends Anchor-pLsr component, repeating the exact same procedure through the assembly of each part.

Thanks!

Throughout rigorous initial interview, webminar introduction to RDP protocol, usability test and final interview, we are grateful to be introduced to expanding assembly methods of synthetic biology. We are also glad that we could provide useful feedbacks to Synbiota, and to watch GEntle software being upgraded from their prototype to current beta version.