The aim of our project was to develop an innovative and cheap detection system for chlamydia trachomatis. Therefore we equipped E. coli bacteria with resistance plasmids containing the genetic information of our mTaz-receptor- and our OmpR/GFP-reporter construct. When working with genetically modified organisms safety of people and environment always needs to be taken into consideration. As our diagnostic method requires direct contact between the patient sample and the detecting bacteria, special provisions needed to be found to ensure an easy but safe handling of the GMOs.
The lab-on-a-chip device
The challenge of our project was to create an application which is safe but still suitable for the requirements in developing countries. Therefore it needs to be handy and easy to use, even for non-scientific stuff, usable without special laboratory equipment, suitable for mass sampling and fast and easy analysable. We met this tasks by encapsulating and immobilizing our bacteria on a lab-on-a-chip device. Firstly the E. coli cells are mixed with 2.5 % alginic acid. During its polymerization the alginic acid develops pores which are big enough to allow smaller molecules like mDAP to enter the capsule but too small for the bacteria to escape. In a next step the biosafety encapsulated bacteria are bonded to the microfluidic diagnostic device. The capsules remain trapped inside the chambers of the device as the size of their diameter (100 µm) exceeds the size of the filter meshes (20 µm) that border the chambers at the openings of the inlet and outlet channels. The diagnostic chamber is probed with the patient's serum which passes through several filters before it is exposed to the capsules and the contained diagnostic bacteria. If the serum contains chlamydia trachomatis the mDAP released by the pathogen is expected to reach the receptors of the immobilized bacteria leading to the expression of GFP. That ensures the reliable diagnosis of chlamydial infections without the risk of a direct contact between people or the environment and the genetically modified E. coli. The chips are solid so that they can be easily transported and handled and are therefore suitable for field projects, as long as the GMO guidelines of the country allow the usage outside of a S1 laboratory.
The alginate capsules
To validate the persistence of the alginate capsules we cultivated beads containing encapsulated bacteria in LB medium (without any selective antibiotics) for several time periods (1h, 2h, 3h). Hourly OD600 measurement of the LB-Medium showed that there was no bacteria escape occurrence.Therefore we postulate that due to efficient fixation of the bacteria in the alginate polymer encapsulation escape is not possible. To validate that the absence of growth was not due to cell death in the encapsulation process we dissolved the capsules in 55 mM sodium citrate for approx. 45 minutes. We analyzed 1ul of the solution through light microscopy and noted cell movement and activity. An increased OD600 after 1 hour of reculturing proved the viability of an uncertain percentage of cells in the capsules. Capsules were as well cracked mechanically and analyzed in the light microscope, where cells were visible in the alginate structures but did not show any movement. We did not measure the power needed to crack the capsules but did need several approaches before we finally managed to crack down the alginate structure to pieces which were small enough for microscopy. Due to our experience with capsule treatment we can assure that breaking of capsules and therefore GMO release will not occur in daily handling of the device. In addition even cracked capsule fragments still fix the GMO’s fierce enough to provide biosafety. To determine lysis of capsules in solution - especially keeping in mind that the pH of vaginal samples which will be needed for chlamydia-testing is about 4.5 to 4.7 we incubated capsules in 100 mL 1 mM HCl and another sample in 100 mL 10 % acetic acid. As expected capsules dissolved quickly in both acidic solutions. After 15 min incubation about 75 % of capsules in the HCl-solution and about 50 % of the capsules in the acetic acid were already dissolved. Both solutions presented a pH of 1. Comparing these solution times to approx. 45 min solution time needed in sodium citrate (pH 7) we do not expect our capsules to lyse during approx. 30 min incubation in vaginal sample in the device. In addition capsule solution time correlates with solution/capsule ratio. We did not perform further testing but expect that lysis of capsules reaches a saturation at one point due to protonation of the used acid.
Risk assessment of our bacteria
Above we have highlighted many aspects of biosafety concerning the release of our bacteria from our device. Additionally, we want to emphasize the biosafety of the used organisms by themselves.
E.coli is a naturally derived organism from humans. JW3367-3 are specially modified bacteria of the strain E.coli that lack the EnvZ/OmpR cascade signalling. We also used the strain DH5alpha. These strains are so called “lab strains” and cannot survive in the human digestive system. They are also not able to produce toxins and are attributed to the risk group one.
Therefore these organisms are classified as the lowest risk for safety and health for humans and to team members and people working with them. Additionally they are not able to survive outside of the laboratory hence do not pose any harm to the environment.
ref. TU Eindhoven 2014
Future steps to maximize biosafety
Taking an outlook into future biosafety maximization one comes up with several approaches. Given the possibility of encapsulating with higher alginate concentrations than 2.5 %, capsule integrity should rise in ratio with alginate concentration. Using other polymerizing agents than alginate should also be taken into account since those could possibly enforce capsule resistivity against acids and other depolymerizing agents. On the other hand biosafety nowadays mainly residues on kill-switch integration in bacterial genomes. Given that there are already plenty of well working established kill-switches available as biobricks, it would be easiest to transform our cells containing the mTaz-sensing and fluorescence reporter system with one of the existing systems. However a light dependent kill-switch would be most efficient and in this case elegant. Given that exposure to light of 495 nm induces fluorescence of the reporter system, light exposure could also induce a light dependent kill-switch. Therefore the device would be inactivated directly after measurement and usage - providing a biosafe waste disposal of the used device.