Overview

The goal of our project is to develop a cheap and convenient way to help farmers and gardeners identify the concentration of various nutrients in soil. Our school, Wisconsin Lutheran College, is located in Wisconsin in the Midwestern part of the U.S. In this part of the country, farming is a major industry and requires good soil to be successful. Soil is an important aspect of plant quality, and unhealthy soil can lead to inconsistency in crop yield and produce health.

To initiate this soil nutrient detection project, we modified the expression of the bla gene which codes for the Beta-lactamase enzyme. This gene is on a plasmid that is contained in Escherichia coli. The expression of the bla gene is controlled by nutrient-specific promoters. While we attempted to create constructs with promoters that regulate the bla gene based on phosphate, nitrate, nitrite, and copper concentrations, our most successful candidate constructs were the ones that regulate based on copper and phosphate concentrations. The bla gene was used as an indicator gene because Beta-lactamase is capable of cleaving a substrate called nitrocefin which will then produce a red color.

Thus, the goal of our project is to provide an easy-to-use kit for farmers or gardeners to use so they can test their soil using an engineered strain of E. coli. This bacterium will produce a red color dependent upon the concentration of a nutrient in the soil. Some of the procedure continues to need refinement but the main steps include: 1) acquire a soil sample, 2) add buffer to the soil sample to create slurry and allow to stand for 1 hour, 3) filter the soil from the buffer, and 4) expose the E. coli containing the nutrient-specific construct and add nitrocefin.

Project Design

Pictured below is our entire construct containing the pcoE promoter controlling the expression of the bla gene. This is in the medium copy number pTwist-Kanr plasmid. When copper is present, the expression of the bla gene increases which results in more Beta-lactamase. The Beta-lactamase will cleave nitrocefin which will result in a red color. Thus, more copper produces more red color.

Pictured below is one of the constructs containing the phoA promoter controlling the expression of the bla gene. This is in the medium copy number pTwist-Kanr plasmid. The phoA promoter negatively regulates the gene it controls when phosphate is present. For this construct, when phosphate is present, phoA reduces the expression of the lacI gene. The lacI gene codes for the Lac repressor. By producing less Lac repressor, there will be more expression from the lac promoter. In this construct, the lac promoter is controlling the expression of the bla gene. So, when phosphate is present, there is more expression of the bla gene which generates more Beta-lactamase and, thus, more red color.

Pictured below is a construct containing the phoA promoter controlling the expression of the bla gene like the construct above except the lacI gene in this construct also has a ssrA tag attached to it. This tag causes the Lac repressor to be degraded more quickly than this protein without the tag. The reasoning behind this is that if phosphate is present, less Lac repressor will be produced but the Lac repressor that is already present in the bacterial cell may continue to cause repression of the lac promoter. By causing the Lac repressor to be less stable with the SsrA tag, the Lac repressor that is already present will be degraded more quickly and potentially produce the desired regulatory response.