Basic Overview

Our parts, as described in the project description, are 3 composite parts made up of 7 of our own basic parts and a number of existing parts. We also helped to improve a part that already existed so that it could better serve our needs.

Our "Search and Destroy" gene circuit was broken up into four main parts:

Figure 1: Native Quorum Sensing Circuit in S. aureus. Image credit: Novick & Geisinger (2008)

Sensing: Detection of AIP (agrAC)

First and foremost, our sense and destroy system must be able to detect AIP. To accomplish this we developed a composite part with the agrAC complex from the quorum sensing operon seen above.

After production of parts BBa_K3083001 and BBa_K3083002 we ran a gel to confirm the existence of the agrAC complex. We expected to see bands on the gel of about 2000 and 4000 base pairs. As can be seen the picture below on the right, we did observe such bands in lanes 2 and 3 for the P2 promoter construct and lanes 6 and 7 for the P3 promoter construct.

Response: Characterization (P2 vs. P3)

For out system to work as desired we must be able to detect the S. aureus colony before it becomes virulent, ie. at a lower threshold of AIP than the native system. By integrating agrAC with different constitutive promoters, P2 and P3, we hoped to determine which promoter allowed for detection of AIP at a lower threshold. This part was also designed to have an RFP reporter below the promoter so that the relative strength of the promoter could be determined.

While we did successfully transform these parts into plasmids as seen above, we were not able to run the desired imaging experiment to quantify promoter strengths due to time constraints. Given more time we would have run a multi-hour incubation experiment with out agrAC parts and used the emission spectra of the RFP over time to parameterize the expression of of the connected agrAC complex.

Sensing: Synthetic Production of AIP (pBAD & agrBD)

To test if our E. coli cells could adequately detect AIP we designed a third composite part to produce AIP in-vitro. We attached the AIP producing portions of the operon, agrBD, to a backbone and then placed it downstream of the pBAD promoter so that it could be induced via arabinose. To ensure it worked properly we also designed the plasmid to have a CFP part after the agrBD sequence so that we could easily confirm its expression.

After production of the part BBa_K3083000 we ran a gel to confirm the existence of the agrBD complex and the pBAD promoter. We expected to see bands on a gel of roughly 3000 base pairs which would indicate a successful transformation. As can bee seen in the gel results above on in the left picture. We did see such a colony on the top series in the 6th lane from the left.

Destroy: Production of Lysostaphin

Once we have successfully detected the colony the next step is to deploy or "kill-mechanism" from the E. coli cells. Instead of designing a new composite part we picked up an existing part designed by the 2012 HIT-Harbin team and improved upon it slightly. We codon optimized it for E. coli and improved the expression of the lysostaphin gene.

We resigned the old part as BBa_K3083004. Once we had successfully transformed it into E. coli plated our resulting colonies on a agar plate with S. aureus to establish a biological kill zone. The plate results can be seen below.

Part Summary Table

<groupparts>iGEM19 CSU_Fort_Collins</groupparts>