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From 2012.igem.org

Design

Indole 3-acetic acid (IAA) is a plant growth hormone that serves as a potent and important auxin to many plants. There are also a variety of synthetic plant growth regulators (PGRs) process the same function as IAA, such as 2,4-D (2,4-dichlorophenoxyacetic acid) and NAA (1-naphthaleneacetic acid). Although auxin is a key factor for plant growth, it can be a metabolic burden to plants at high concentrations and prohibit plant growth, which contrasts its initial goal.

 

Imperial College London 2011 iGME team claimed that molecules of IAA are chemically labile in aqueous solution, so they produce IAA in soil to promote rather than stunt plant root growth. They also regulated output of IAA through change However, root sensitivity to IAA differs from variety to variety. There is no guarantee that E.coli which produces IAA will fit in all situation. Our designed RNA riboscaffold makes it possible to change the output of IAA in a range to adapt the need of plant root growth.

 

Coexpression of enzymes IaaM and IaaH catalyzes the reduction of tryptophan to IAA. We plan to use this system to assess the potential of RNA scaffold D0 to accelerate reaction speed and our RNA riboscaffolds to regulate reaction speed. We fused IaaH to a single copy of PP7 (IaaH-2X-PP7) and IaaM to a dimer of MS2 (IaaM-2X-MS2) with overlap extension PCR procedure.

 

First goal: accelerate reaction speed with RNA scaffold D0

 

 

Two enzymes related to the reaction are fused to RNA scaffold D0 to get spatial organized.

 

Second goal: regulate reaction speed with RNA riboscaffold D0

 

 

The interaction inhibits the binding function of MS2 aptamer in the absence of theophylline. IaaM-2X-MS2 is dissociative in cell and the speed of reaction is normal.

 

 

When theophylline is added, the fold of the loop is changed and thus the interaction will disappear, leading to the binding of MS2 aptamer and corresponding protein. IaaM and IaaH will get closer to accelerate reaction speed.

 

Test:

 

 

We plan to determine the concentration of IAA with salkowski assay. The standard curve has been made with IAA in LB.

 

Plasmids for co-expression:

 

pZCM

 

 

pZCH