Part:BBa_K5380001:Experience

Arabidopsis Thaliana utilizes NIN-Like Proteins (NLPs), which undergo conformational changes in the presence of Nitrate (Konishi et al., 2013; Chardin et al., 2014). NLPs, having gone aforementioned conformations, will interact as transcription factors by binding to Nitrate Responsive Cis-Elements (NREs), thus increasing expression rates of downstream elements (Konishi et al., 2013; Chardin et al., 2014).

We used this part in our project as a promoter to up regulate downstream genes (in our case RUBY or GFP) in response to the presence of nitrate in our plant system. We were unable to verify its efficacy in our plant, as we were unable to verify the insertion of the part into our plasmid backbone, but its efficacy is well validated in previous publications. We theorize expression of genes attached to this promoter to scale roughly linearly to the presence of nitrate in plant material, and it is unknown whether or not it would function properly outside of a plant system, though both of these ideas require further validation.

This NRE system was difficult to synthesize traditionally due to repetitive regions in the DNA. Primer design also required additional attention due to issues with melting temperatures. As a result, this NRE system was synthesized using enzymatic DNA synthesis through a special vendor (Molecular Assemblies).

We theorized that use of NREs with a RUBY reporter system can potentially be tuned to create binary expression at specific thresholds of nitrate concentrations by using DODA Decarboxylase (BBa_K5380007) to prevent the rate limiting step of forming betalamic acid from DODA until expression thresholds are high enough, but this is yet unvalidated, as we were unable to verify the insertion of the part into our plasmid backbone.

Applications of BBa_K5380001

NREs can be used in soil nitrate diagnostics in Arabidopsis thaliana by being coupled to colorimetric or fluorescent reporters to cause plants to change colors or fluoresce based on soil nitrate levels.

NREs can be used in bioremediation in Chlamydomonas reinhardtii as a way of up-regulating proteins for processing excess nitrates upon increases in nitrate concentrations, allowing for increased expression only during times when it is necessary, enabling the algae to have decreased expressive strain when unnecessary.

As a more general rule, it can serve as a genetic switch for any system in which a response to nitrate is required, effectively making attached genes roughly nitrate-activated.

We theorize that use of NREs with a RUBY reporter system can potentially be tuned to create binary expression at specific thresholds of nitrate concentrations by using DODA Decarboxylase (BBa_K5380007) to prevent the rate limiting step of forming betalamic acid from DODA until expression thresholds are high enough, but this is yet unvalidated.

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