This video demonstrates a method to culture and dispense fluorescent strains of CL gans for high throughput screening of chemical libraries or detection of environmental contaminants to assess the effect of chemical or environmental agents on specific protein activity. In CL elgan, bacterial cultures are first prepared as food for the worms and GFP transgenic. Synchronized worms are cultured.
0.5 to 2 million worms are then collected, washed and dispensed into 3 84. Well microtiter plates, then small molecules from a chemical library or test samples such as water, food, or soil are added to the worms. Finally, the fluorescence of the worms is measured with a microplate reader.
Data analysis of fluorescence intensities reveal small molecules that modulate inducible transcriptional pathways or allows for the detection of environmental contaminants. The main advantage of this technique over existing methods, like in vitro and cell culture based assays, is that time will not be wasted on compounds that are toxic or inactive in vivo. This high throughput method can help identify small molecules that modulate diverse inducible pathways.
Though we developed this method for high throughput screening of small molecule libraries, it can also be applied to biosensing of contaminants in food and environmental samples. Demonstrating the procedure will be Andrew Dine, a technician for my laboratory and Q long a postdoc for my laboratory. Prepare bacterial worm food ahead of time.
Begin by inoculating 500 milliliters of terrific broth, supplemented with 50 micrograms per milliliter of streptomycin with five milliliters of saturated e coli. OP 50. Place the culture on a shaker and grow overnight at 37 degrees Celsius.
The next day, split the culture into 10 50 milliliter tubes. Then place the tubes in a refrigerated centrifuge and spin at 2, 500 RCF for 20 minutes. Following the spin Resus, suspend each bacterial pellet in 10 milliliters of liquid nematode growth media or NGM.
Shake the suspensions horizontally. A floor shaker for 15 minutes to resuspend the bacteria. Next, centrifuge the bacterial culture at 2, 500 RCF at four degrees Celsius for 20 minutes.
Decant the NGM and weigh the bacterial pellet. Then add an equal volume of NGM buffer and resuspend the pellets by vortexing. Finally, aliquot three milliliters of the resuspended bacterial culture into 15 milliliter tubes and store them at minus 20 degrees Celsius until they are needed.
In this demonstration, the effect of the stress inducing Xeno Duke loan on the transcription factor Sskn one will be measured as an example. Sskn one is known to activate cytoprotective genes during oxidative and xenobi stress. A large scale culture of the Trenchgenic c gans line VP 5 96, which carries two fluorescent constructs is prepared.
VP 5 96 worms express both the GST four promoter driving GFP, which is used to monitor the activity of the transcription factor. Sskn one and the DOP three promoter driving. RFP is used as a standard for worm number normalization.
DOP three is the promoter for a dopamine receptor and is constitutively expressed in neurons throughout the body. In the example, given the xenobi lon strongly activates sskn one and increases the amount of GFP expressed from the GS T four promoter to prepare cultures begin by combining 150 milliliters of N NGM buffer, 1.5 milliliters of LB 150, microliters of one molar cholesterol and 75 microliters of 100 milligrams per milliliter.Streptomycin. Adding the LB helps to prevent the worms from sticking to the glassware and plasticware, but this amount is not enough to support bacterial growth.
Filter to sterilize the mixture, then transfer it to a sterile one liter flask and set the flask aside to release the eggs from GR adult worms. Wash the bacteria from the worms by centrifuging them and replacing the NGM medium until the supinate is clear. Wash the worms a second time by Resus suspending them in sterile water centrifuge again following the spin.
Remove the supinate and fill the tube with 3.75 milliliters of sterile water. Add one milliliter of household bleach and 250 microliters of 10 normal sodium hydroxide mixed immediately by inversion five to six times. Place the tube in a rotator after the worms have been exposed to bleach for four minutes.
Place the tube under a stereo microscope. Most of the adults should be lies. Shake the tube vigorously 15 to 20 times to help lies any remaining adult worms after lysis has occurred for a total of five minutes, add sterile water until the tube is full.
Mix by inversion five to six times to collect the eggs. Centrifuge the tube for one minute at 500 RCF. Wash the eggs three times by Resus, bending them in 10 milliliters of sterile water, spinning and removing the supinate.
Next, dilute a 100 microliter sample of the eggs 100 fold by adding 10 milliliters of NGM buffer. Then pipette three five microliter aliquots onto a Petri dish lid under a microscope. Count the number of eggs in three separate five microliter aliquots.
To estimate the total number of eggs. Add 200, 000 to 2 million eggs to the flask with NGM buffer and shake the culture at 100 RPM at 20 degrees Celsius. At least 16 hours after the eggs have been added to the flask, uses sterile serological pipette to remove approximately 0.5 milliliters of the suspended worm culture.
Pipette three five microliter drops onto the sterile lid of a Petri dish. Place the lid in a minus 20 degrees freezer for one to two minutes to paralyze the worms once the worms have been paralyzed. Count the average number of live hatched worms per five microliters with a stereo microscope to estimate the total number.
Thaw the frozen OP 50 bacterial culture prepared earlier by placing it on a bench for a few minutes. Then add three milliliters of 50%OP 50 per 500, 000 hatched worms. Shake the flask 100 RPM at 20 degrees Celsius.
The worms will develop into L four larvae and young adult stages in about 51 hours. Monitor the amount of bacteria throughout the incubation by visual inspection. Add more bacteria if the media becomes clear.
Use a sterile serological pipette to transfer approximately 0.5 milliliters of the suspended worm culture to a standard NGM agar plate. View the worms with a stereo microscope to make sure that most are L four larvae and young adults under brightfield microscopy, L four larvae will have a ventral clear spot in the middle of the body. Young adults will be slightly larger and will not have a clear spot.
Next, pull the worm culture into sterile 50 milliliter tubes. Place the tubes in a test tube rack in the hood and allow them to settle unhatched eggs or worms that do not develop. Settle more slowly than developed worms.
After 10 minutes, remove the supinate, which contains unhatched and undeveloped worms by aspiration at approximately 10 milliliters of NGM buffer to each pellet and suspend the worms by gently swirling the tubes. Collect all of the worms into a single 50 milliliter tube. Spin in the swinging bucket centrifuge at 500 RCF for 30 seconds.
Then resuspend the worms in 50 milliliters of NGM with 1%lb to wash them. Repeat this, wash three to four times to remove the bacteria after washing. Fill the tube with NGM plus LB buffer to 50 milliliters.
Pour this into a custom worm dispensing flask at a stir bar and place it on a stir plate. A minimum of about 20 milliliters or 60, 000 worms is needed to fill the dead space at the dispensing flask and dispenser cassette. Count the number of worms in three five microliter drops as previously shown.
After counting at NGM plus LB to a concentration of 12 to 15 worms per five microliter drop each 3 84 well plate requires about 35, 000 worms or 11.5 milliliters of suspended worms. Load a 10 microliter dispensing cassette into the dispenser and sterilize it by priming with 70%ethanol. Rinse out the ethanol by priming with sterile water.
Insert the end of the dispensing cassette into the flask so that it is just above the stir bar without disrupting its movement. Make sure that the worms remain suspended throughout the flask. Program the dispenser to dispense worms at low speed with two pre pulses prime and run At least 10 milliliters of suspended worms.
Fill the microtiter plates, which should already contain test compounds rapidly to prevent the worms from settling in the tubing. Here the test compound previously loaded onto the microtiter plate is due.Glow. Seal the plates with breathable tape to prevent dust from collecting in the wells.
Place the plates on a shaking platform in an incubator at the appropriate temperature for the assay. Do not stack the plates as this can restrict the flow of air. Place the target plate into a microplate reader to measure the fluorescence intensity of each well with the appropriate emission and excitation wavelengths here.
The filters used for GFP are excitation at 4 85 and emission at 5 28. The filters for RFP are excitation at five 40 and emission at five 90. After reading the plate, calculate the ratio of GFP to RFP and normalize with the readings of the control wells, which do not contain the activating compound to determine the exact fold difference of the fluorescence intensity derived from individual treatments.
The SSKN one assay shown here uses a chromosomally integrated dual report, a strain when the worms were manually counted with a stereo microscope. The number of worms correlated with the volume dispensed with an R squared value of 0.94 and a p value of less than 0.0001. This indicates that the number of worms dispensed into each well can be controlled by adjusting the volume dispensed.
The total GFP fluorescence per well shown on the left and RFP fluorescence per well shown on the right is highly reproducible from well to well across a 3 84 well plate in these figures. XS indicate the relative fluorescence measured in each well. Solid lines indicate the means and broken lines indicate three standard deviations above or below the mean fluorescence of all wells had a coefficient of variation below 9%As shown in this figure, GFP fluorescence from the GST four promoter is linearly correlated to RFP fluorescence from the DOP three promoter across 3 84 wells with an R squared value of 0.62 and a p value of less than 0.0001.
Therefore, the non inducible RFP reporter can be used to normalize the inducible. GFP reporter. When expresses a ratio of GFP over RFP fluorescence becomes highly reproducible from well to well across a 3 84 well plate.
Calculating the ratio of GFP to RFP reduced the coefficient of variation to below 6%demonstrating the ability of the DOP three promoter RFP reporter to reduce variability as shown here. The induction of GFP relative to RFP with an SK N one activating xeno biotic is robust and highly reproducible across a 3 84 well plate, the mean relative fluorescence ratios of all control and due GLO wells are marked with solid lines. Three standard deviations above the control mean and below the dlo mean are marked with broken lines.
Once mastered, starting a worm culture can be done about one hour on day one and collection and dispensing. A worms can be done about two hours if done properly. While attempting this procedure is important to remember to start with at least 60, 000 worms and keep them suspended while dispensing, following this procedure screens with other, for instance, report strings for other inducible pathways can be performed in order to develop modulators for other pathways After its development.
This technique can pave the way for researchers in the fields of high throughput screening and and biosensing to rapidly identify small molecule pathway modulators or detect environmental contaminants. After watching this video, you should have a good understanding of how to culture dispense and measure fluorescent elegan strains for high throughput screening if inducible transcriptional pathways or testing multiple samples for environmental contaminants.
