Project
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Project Inspiration
Many of us have faced the problem of dairy spoilage in our daily lives. Standing before the refrigerator, we are conflicted by the clean, creamy scent of a gallon of milk and the passed expiration date on the lid. Unfortunately, properly estimating the safety of dairy products can be difficult for consumers, as well as for distributors that handle billions of gallons of milk per year. As environmentally conscious citizens, we worry about the impact of food waste and increased factory farming. And so, as scientists and engineers, we worked toward a solution. We considered how we as individuals could work to combat this problem and what approaches would be most meaningful in their application. Inspired by a number of other biosensors including IIT Kharagpur's 2015 Project and the structure of the Lac operons, we are excited to introduce a simple, cell-free testing platform for AHL, a quorum sensing molecule released by spoilage bacteria.
Project Description
Expiration dates on food help consumers ensure quality and safety. Although the ‘best-by’ dates reflect products at its best nutritional value and freshness, they do not actually mean that products are spoiled or unedible after the date. However, many people, not wanting to risk potentially bad food, often times throw it away leading to a substantial amount of food waste. According to the United Nations Food and Agriculture Organization, one third (1.3 billions tons) of the global food supply is wasted. In hopes to reduce food waste, we designed a biosensor to detect food spoilage, specifically in milk. Inspired by IIT Kharagpur Team’s project in 2015, we engineered E.coli to detect autoinducers, acyl-homoserine lactone (AHL), that is released by food spoiling bacteria. AHLs are intercellular signalling molecules released by gram-negative bacteria to regulate gene expression based on population density. It is understood that as food spoiling bacterial population increases, the amount of AHL increases; therefore, a high level of AHL can be an indicator of food spoilage. We took advantage of the lux operon, originated from Vibro fischeri, to detect the amount of AHL in milk using quorum sensing. According to Ammor, Michaelidis, and Nychas (2008), as AHL molecules accumulate to reach the threshold concentration, luxR response regulator will activate the transcription of its downstream operon which, in this case, is designed to be reporter pigment genes. In other words, detection of high AHL level is visualized by the expression of pigment proteins, allowing us to determine if spoilage has occurred while ensuring food safety by using a cell-free expression system. Additionally, to provide convenience to customers, the detection system is lyophilized on a paper as a paper-based, cell-free biosensor.
Background information
Our device utilizes quorum sensing to determine whether or not bacteria are present in milk and if so, what quantity they are present in. Quorum sensing takes advantage of bacterial intracellular signaling by detecting molecules that bacteria send to each other to signal their existence. Gram- negative bacteria excrete a molecule called acyl-homoserine lactone (AHL) into their extracellular environment, which is an autoinducer that nearby bacteria can detect. As the concentration of bacteria increases, the amount of detectable AHL also increases, so that bacteria can use AHL concentrations to know whether or not there are other bacteria near them. Bacteria use this information to know if they should express certain genes, including the one that produces more AHL. Because AHL is expressed in a positive feedback loop, as more of it enters a bacteria, more is produced by the bacteria. This means that there is an exponential relationship between the amount of bacteria in a substance and the amount of AHL that can be detected. Furthermore, because bacteria detect AHL in nature and change gene expression based on its presence, engineering a part to detect AHL was relatively straightforward.
A critical operon in quorum sensing is called lux operon. Lux operons are under the control of quorum sensing. All lux operons have a consensus organization of luxCDAB(F)E genes. LuxA and luxB genes encodes for luciferase while luxC, luxD, and luxE encode for the fatty acid reductase complex that is involved in luminescence reaction. The lux operon is activated by a complex composed of the transcription factor, luxR protein, and the quorum sensing molecule, AHL.
Parts Description
BBa_K3251000 is an improvement of Biobrick BBa_F2621, consisting of the luxR protein coding region, as well as the luxpR promoter which can be activated by the binding of luxR+3OC6HSL complex. Part BBa_F2621 was shown to be defective since it lacks the BBa_R0063 promoter (according to iGEM11_Wageningen). We added an inducible IPTG promoter upstream to the luxR coding region and a yellow chromoprotein downstream of the luxR gene. This new part construct allows the inducible expression of luxR by IPTG with a visualization by the yellow reporter chromoprotein.
This part includes an addition of a second chromoprotein, BBa_E1010, to the biobrick BBa_K3251000. This red reporter protein serves to visualize the level of 3OC6HSL. In the presence of IPTG, the LuxR gene encodes for the LuxR protein and the yellow chromoprotein, which serves as a control to ensure the detection system functions properly. Binding of the LuxR+3OC6HSL complex will activate the luxpR promoter, which allows the expression of the red chromoprotein. Depending on the concentration of 3OC6HSL, the combination of the red and the yellow chromoproteins will be visualized with a color gradient ranging from yellow to red.
Our goals: We would like to detect milk spoilage by detecting the presence of quorum sensing molecules(AHLs), using a paper-based, cell-free expression system.
How it works:
The plasmid
Our plasmid construct is built upon the Lux operon, which is widely used in bacterial quorum sensing. The most studied Lux operon is originated from vibrio fischeri and encodes genes for self-regulation and the production of luminescent proteins. The first component of our construct is an inducible IPTG promoter that activates the expression of a yellow chromoprotein. The yellow chromoprotein serves as a control to ensure the detection system works correctly. A red chromoprotein is downstream of the luxR gene and its expression is promoted by the pluxR promoter, which is activated by the 3OC6HSL+luxR complex. Therefore, when IPTG and 3-oxo-C6 homoserine lactone (3OC6HSL) are present, the construct will produce both yellow and red chromoproteins, creating a color gradient ranging from yellow to red depending on the level of 3OC6HSL in the environment--a higher intensity of red color indicates a higher level of 3OC6HSL.
Cell-free system
Our construct serves to detect and visualize milk spoilage through the fusion of LuxR gene and two chromoproteins. The construct is cloned into a vector and transformed into bacteria. However, it is not appropriate for the general public to use living cells as a food spoilage detection device. Engineered bacteria extract is thus lyophilized to a piece of paper. Not only can the construct be expressed in a cell-free paper-based system, the safety of users is also prioritized.
Lyophilization onto paper strip
We hope to lyophilize our construct onto paper for easy application by consumers. The concept of paper strips is very similar to pH test strips. The cell-free extract that contains our construct, protein machinery, and any ingredients necessary for protein synthesis will be freeze-dried to the paper strip. The paper strip can be activated by adding a few droplets of the milk sample. After a few minutes of hydrating the strip, we should be able to visualize a color that would range from yellow to red, which is indicative of the amount of AHL present in the milk sample. The higher the AHL concentration, the more intense the red chromoprotein will appear.