Team:Stanford/Composite Part

Stanford iGEM Team Wiki

Composite Parts

A Table of All Composite Parts

Part Name
1 BBa_K3258017 Best New Composite Part: Self-Amplifying Fluorescent RNA (MDV-Q-beta-Spinach)
2 BBa_K3258006 Infection Reporter (Design Using BBa_K2832003)
3 BBa_K3258008 M13 Helper Plasmid for PREDCEL+
4 BBa_K3258009 Selection Plasmid PREDCEL+
5 BBa_K3258010 T7 to T3 Selection Schema PREDCEL+
6 BBa_K3258011 lacI-derived biosensor selection schema (positive selection) PREDCEL+
7 BBa_K3258012 lacI-derived biosensor selection schema (negative selection) PREDCEL+
8 BBa_K3258013 Selection Plasmid T7 to T3 PREDCEL+
9 BBa_K3258014 Selection Plasmid T7 to T3 PREDCEL+ (Alternate Version)
10 BBa_K3258018 Q-beta EFTu-EFTs fusion protein
11 BBa_K3258025 MDV + Ampicillin with 1 Stop Codon
12 BBa_K3258026 MDV + Ampicillin with 2 Stop Codons
13 BBa_K3258028 MDV + CAM Flipped
14 BBa_K3258029 MDV + CAM Flipped with Terminator
15 BBa_K3258030 MDV + Qbeta + CAM Resistance Gene
16 BBa_K3258032 MDV + GFP Flipped
17 BBa_K3258033 MDV + Qbeta + Spinach Aptamer Plasmid
18 BBa_K3258034 T3 Promoter + CAM Resistance Circuit

Best New Composite Parts

We submit Self-Amplifying Fluorescent RNA (MDV-Q-beta-Spinach) (BBa_K3258017) as our best new composite part. This is a part composite composed of self-encoded Qbeta replicase with a Spinach aptamer attached, both sequences flanked by midivariant regions recognized by Qbeta, referred to as MDV regions upstream and downstream. When expressed in E. coli cell free extract supplemented with DFHBI, Spinach fluorescence increases due to the replication of the RNA by the Qbeta replicase enzyme. This creates a self replicative cycle, and the amplification of the Qbeta and Spinach aptamer.

Testing In Vitro

Team Pic

The functionality and operation of the MDV-Qbeta-Spinach construct was tested in Sigma 70 myTXTL Cell Free Cell Lysate. Initial tests were conducted comparing fluorescent output from another construct, mRFP-Spinach, with the fluorescent output of MDV-Qbeta-Spinach. Trials were conducted running reactions with each construct at the same time at 29 degrees Celsius for 18 hours and measuring fluorescent output. Results showed significant differences in fluorescent output between MDV-Qbeta-Spinach and mRFP-Spinach, suggesting that MDV-Qbeta-Spinach was operational in vitro in the used cell free lysate.

Testing MDV Region Impact on Replicability of Qbeta System

Team Pic

After confirming the functionality of the MDV-Qbeta-Spinach construct with in vitro experimentation, the impact of the MDV regions was tested. Each MDV region (BBa_K3258015 and BBa_K3258016), or midivariant region, is a RNA sequence experimentally classified in previous literature to be a good substrate for Qbeta binding and replication. Flanking a given gene of interest, especially Qbeta, with the midivariants should make the whole sequence more conducive to replication and amplification by Qbeta. This was tested by comparing the fluorescent output of the MDV-Qbeta-Spinach construct in vitro with trials of a Qbeta-Spinach only construct, which lacked the MDV flanking regions. Run in triplicate, the results suggested a clear improvement of the MDV flanked Qbeta-Spinach over the control Qbeta-Spinach only, with no trial of the MDV-Qbeta-Spinach dipping below the highest performing trial of the Qbeta-Spinach. The differences in amplification of each construct suggested a ~30% improvement in the amplification rate with the MDV flanked construct versus the non-MDV flanked construct.