Difference between revisions of "Part:BBa K3258016"

Latest revision as of 20:09, 21 October 2019

Downstream MDV

Midivariant RNA (MDV) is RNA that is readily recognized and replicated by Q-beta replicase, an RNA-dependent RNA polymerase. To increase the replicability of RNA by Q-beta replicase, the MDV can be split into sub-parts and added upstream and downstream of the RNA of interest. This is the downstream complementary DNA that should be added to the cDNA of interest.

MDV Regions with Qbeta Protein In Vitro

In order to test whether the MDV regions allowed for self replication, especially for in vitro testing, a construct was designed featuring MDV with the Qbeta subunit as well as the spinach aptamer. Qbeta had previously been demonstrated to be capable of self replication along with the midivariant regions, making this a good control case. If Qbeta was able to self replicate with the MDV regions as expected, then fluorescence should increase as a result of the spinach aptamer tag added to the Qbeta subunit RNA. The functionality and operation of a 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

After confirming the functionality of the MDV-Qbeta-Spinach construct with in vitro experimentation, the more general impact of the MDV regions for affecting the replicability of a construct was tested. Hypothesizing that 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 trials of 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.

MDV Regions with Genes Beyond Qbeta

After demonstrating that the MDV regions allowed Qbeta self replication and were an important factor to the replicability of the Qbeta construct, the MDV regions were then applied to other target genes. The replicability of GFP RNA with and without MDV regions when exposed to Qbeta was determined in vitro, with a Qbeta construct featuring just the subunit as well as the full linked fusion protein. As shown in this data, GFP flanked by the MDV regions, in both the Qbeta full linker construct as well as with the subunit alone, were both shown to undergo replication with Qbeta in vitro.

MDV Regions with T7 Polymerase Gene for T7 Polymerase T3 Promoter Recognition Evolution

Finally, once the feasibility of making non-Qbeta genes replicable and amplifiable by the MDV regions was demonstrated, the MDV regions were applied to the T7 Polymerase gene as an application of the Qbeta based DiCE method of directed evolution in vitro. By adding MDV regions to the T7 Polymerase, the T7 Polymerase was made replicable by Qbeta, which is a highly error-prone RNA transcriptase. This introduces mutations into each round of replication, which when put under selective pressure and linked to the replicability or availability of Qbeta in a system, should result in the accumulation of mutations that favor more well adapted candidates to the environment. In this system, the goal is the evolution of a T7 polymerase capable of binding to a T3 promoter region, and the selective pressure is introduced by linking Qbeta availability, which is responsible for replication, to binding and transcribing a T3 promoter. After 5 rounds of evolution, which involve passaging and expression inside cell free solution in increments, a ~400% increase was demonstrated in the rates at which T7 polymerase resulted in the expression of fluorescent reporter protein underneath T3 promoter regions with round 5 RNA compared to control non-evolved stock RNA for the polymerase. This suggests the feasibility of DiCE in vitro evolution as well as the usage of MDV regions for creating sequences replicable by Qbeta.

Sequence and Features