Whole-cell biosensors offer numerous advantages over conventional detection methods due to their cost-effectiveness, renewability, and environmental friendliness. As a result, they have attracted considerable interest in the field of synthetic biology, making them an attractive focus for iGEM projects. Our project provides a robust application platform for biosensors.
The paper strip approach allows for easy use without any kind of laboratory equiptment for the readout for any biosensor with a β-Galactosidase as reporter gene. The biosensor could even be used in households!
The protocol for paper strips is as follows:
Inoculate a 25 ml LB overnight culture with one clone containing the biosensor and
the
according
antibiotic for selection and let grow at 37°C, 180 rpm for about 16 h. Harvest the
cells
by
centrifugation at 3500 xg for 15 min, discard the supernatant and resuspend the cell
pellet in 1 mL
sterile, preheated (37°C) drying protectant solution (0.5% (w/v) peptone, 0.3%
meatextract, 10%
gelatine, 1% sodium ascorbate, 5% raffinose, 5% sodium glutamate). Premark cut
Whatman
filter paper
pieces with a pencil spot and pipet two times 5 µl of the cell suspension onto the
spot.
The paper
strips then can be placed in a 50 ml falcon with a holey lid and lyophylized over
night.
After
lyophilisation, the paper strips can be stored in the fridge.
To test the paper strips, they can be placed in a petri dish. To reactivate the bacteria, 10 µl of LB-medium need to be pipetted on the premarked spot. Afterwards, 10 µl of the testing solution and 10 µl X-Gal (1 mg/ml in H2O) are pipetted on the spot. The paperstips are then incubated in the dark to prevent a reaction of X-Gal with light at 37°C for about 30 minutes. If the test is positive, blue color is developed on the paper strip.
The boba system can be used for any biosensor that uses a fluorescent protein as a reporter gene and allows for 24/7 monitoring.
The protocol for the bobas is as follows:
An overnight LB-culture containing the biosensor and a 2.5% alginate solution are mixed
in the ratio
of 4:1 and are drawn through a cannula into a needle. The solution is then dropped
slowly into a
petri dish containing a 0.1 M CaCl2 solution. The bobas then can be transferred into the
testing
solution and fluorescence can be observed with a fluorescent microscope wit a GFP
filter.
At the beginning of the project we needed to ampify the plasmids we recieved. The retransformations were done with chemically competent DH10ß cells, the 5 ml overnight culture grew for 8 h at 37°C, 180 rpm and the plasmids were purified using the ROTI®Prep Plasmid MINI-XL kit. The obtained plasmid concentrations were all below 30 ng/µl and thus too low for sequencing or consequent cloning steps. For the next attempt, we grew the cultures over night but still the concentrations were too low. So we decided to change from DH10ß to DH5α cells but still the plasmid yields remained too low. Simultaniously we wanted to switch from the golden gate cloning system, which uses blue white screening to one which uses a GFP gene being cut out as a screening system. For this system, Top10 cells were recommended for cloning, so we again changed to Top10 cells. After again obtaining low plasmid yields with Top10 cells, we tried to purify plasmids from the same overnight culture with two different kits and noticed that we obtained higher plasmid yields with the NucleoSpin® Plasmid EasyPure kit than with the ROTI®Prep Plasmid MINI-XL kit and therefore changed kits. So we used Top10 cells for the plasmid amplification and the NucleoSpin® Plasmid EasyPure kit for purification. We also noticed that with this combination we would usually get better yields by growing the culture for 8 h than growing over night at 37°C, 180 rpm. Consequently we always tried to inoculate the 5 ml overnight culture in the morning and prep the plasmids in the evening. If that wasn't possible, we grew the culture over night at 30°C, 180 rpm.