Project Description

Save our L.A.B.

The present day dairy industry heavily relies on the use of lactic acid bacteria like Lactococcus lactis. However these bacteria are particularly vulnerable to bacteriophages. And phage infections can lead to lysis of the cell culture which leads to a downgraded or even unsellable product. In the worst cases the infected milk is discarded entirely. This results in huge financial losses, stress on staff and problems in obtaining a consistent quality product. So preventing phages infections within dairy production culture is essential in the present day industry. Currently multiplex PCR is used to detect the presence of common phages species, which require regular sampling. The aim of this year’s iGEM project is to simplify this detection and make it more continuous by linking a CRISPR detection system to a fluorescent signal and in parallel a restriction fragment length polymorphism analysis. Our systems works in several steps, which are explained in more detail below.

• Infection: Since our system is built in the same organism as used for the fermentation, phages will infect our system automatically.
• Spacer acquisition: After infection the CRISPR/Cas complex will recognize the phage DNA and harvest a 25-30 nt spacer and incorporate it into the CRISPR array.
• Specific nuclease: The phage specific spacer is turned into a guide RNA which together with Cas9 forms a nuclease that targets a sequence specific for the phage.
• Signal: Our signalling plasmid contains a sequence homologous to the phage DNA in a repressor/promotor region of a GFP gene. Cutting by Cas9 leads to either expression or loss of expression of GFP, which gives an easy to detect signal.
• Multiplexed: If our signalling plasmid would contain multiple sequences homologous to different phages we can detect and distinguish multiple phages. We want to do this by incorporating restriction enzyme recognition sites around our phage sequences. Cutting of these sites will result in a change of restriction pattern after a restriction analysis.

We think dairy industry would benefit most from the real-time detection of the 10 most common phages using the GFP signal, since they have the means and knowledge to perform a multiplexed PCR to determine the type of infection after getting the signal. However we think our system can also be used for other applications where they don’t have the money and means to carry out multiplexed PCR.