Our project is to make circuits that express chromoproteins when heme is present. To do this, we used a mechanism in which bacteria respond to heme to make an efflux pump. In this system, HssS and HssR were used to recognize from the outside of the cell and HrtR to recognize from the inside.
1. HrtR System
HrtR is a self-regulator that binds directly to the promoter’s palindrome sequence(ATGACACAGTGTCAT) in front of its CDS. When it binds to the promoter, it acts as an inhibitor that prevents the transcription of itself.
We ordered the hrtR sequence of the gram-positive Lactococcus lactis in IDT and cloned it into DH5alpha using pSB1C3 and TA cloning. In the picture above, the white colonies were inserted with the HrtR and the RFP was deleted. The red one is that the RFP still exists because HrtR is not inserted normally.
Commonly used GFPs must be exposed to ultraviolet light in order to see strong green color. So we planned to use chromoprotein instead of fluorescent protein because we thought that fluorescent proteins would be difficult to use in common life. The 2013 Uppsala team's project was using Lactobacillus and chromoprotein, and we decided to use these parts. This photograph shows the expression of their amilCP in E. coli DH5alpha. AmilCP will be more noticeable because it has a blue color that is complementary to the color of the feces.
Our final design using above system is as follows. HrtR is expressed in cells at a constant concentration by negative feedback. When HtrR binds to heme, its inhibitor function is lost and transcription occurs. Therefore, the concentration of chromoprotein increases and the color of the bacteria in the feces becomes darker.