The overall goal of this procedure is to identify cellulase expressing clones from a metagenomic library. This is accomplished by first replicating a previously made metagenomic library using a robot following incubation of the replicated library at 37 degrees Celsius for 24 hours. The growth of the e coli clones is measured, lysis, buffer, and substrate is then added to the replicated library.
Ultimately, results can be obtained that shows cellulase activity through colormetric absorbance measurements using a plate reader. Hi, I'm Keith from the laboratory of Steven Hallam in the Department of Microbiology and Immunology at the University of British Columbia. Today we will show you a procedure for the high throughput functional screening of metagenomic libraries.
We use this procedure in our laboratory to study biomin cellulase activity from metagenomic libraries. So let's get started. Before starting this protocol, prepare a metagenomic library stored in a 384 well plate format.
This study used the PCC one copy control SMID vector in combination with Farge T one resistant TRANSFECT max EPI 300 T one resistant e coli cells as the library host to start this procedure, remove the plates containing the library from minus 80 degrees Celsius and defrost them at 37 degrees Celsius for approximately 20 minutes. Meanwhile, use the UV light sterilizing feature on the QIX two to sterilize the robot for 15 minutes. Set up the Q fill three as per manufacturer's instructions with the media bottle attached to the manifold via the sterile tubing program.
The Q fill three for the appropriate amount of media and set it to fill a 45 microliter volume. In each, well purge the air from the tubing and manifold using the purge feature of the robot until media is visible. At each pin of the manifold, fill the desired number of plates with LB media using the Q fill three.
Each plate takes approximately 20 seconds to fill. Next, load the library plates and the fresh plates into the appropriate areas of the Q picks. To robot fill the cleaning baths with the appropriate reagents.
2%micro 90 in the rear bath, autoclave distilled water in the middle bath, and 80%ethanol in the front bath. Using the replicating program of the qix two, select the appropriate head as well as the number and types of both source and destination plates. Also set up the head to clean between replications.
Usually six cycles in each bath is enough. The capacity of the machine is 10 plates at a time. Set the machine to run.
It'll take approximately 15 minutes to replicate it. Full capacity with a 384 pin head. Once the plates are replicated, grow the plates at 37 degrees Celsius for 24 hours in a humidity box.
The long incubation time is necessary since a high copy number of smid is being induced with the addition of arabinose. Meanwhile, clean the Q fill three robot as described in the written protocol. Following incubation, remove the plates from the 37 degrees Celsius incubator.
Set aside the lids and place the plates onto the magazine loading platform provided with the rapid stack. Be sure to keep track of the order of the plates as the software does not. The plates at the bottom of the stack will be read.
First, remove one magazine from the rapid stack. Ensure it is empty and push it onto the stack of plates to be read. Grab the magazine by the handle and the plate should load into the magazine.
Load the magazine into the rapid stack in the correct orientation. Open the scan its re program on the connected computer. Select new session and name it appropriately.
Choose calling flat bottom 384 Well plate as the plate. Type in the plate layout area. Select the wizard button and choose it to add 384 unknowns to the plate.
In the protocol area, select the well loop option and enter for 384 wells. A well loop icon will appear in the flow tree on the left hand side of the screen. Select the icon and click to add photometric measurement.
Set it to read at 600 nanometers. Save the protocol with a unique name and close the scan its re program. Then open the polara RS program.
On the main page is a table listing the instruments connected to the rapid stack device under the VARI scan heading, click the run session option. The assay window should then appear with a flow chart in the middle. Select the run session item, then find the name of the saved scan.
Its re run in the dropdown menu that appears on the right side of the screen. Once the protocol has been selected, click run. This assay a box will pop up asking which magazine to use as the source and which magazine is empty.
The lower box on the screen corresponds to the front magazine zine. Ensure that both the rapid stack and the Vario scan are turned on and click start. The rapid stack will automatically load the plates into the plate reader allowing for continuous measurements of the plates.
Observe the first plate loading into the Vario scan to ensure proper alignment of the machines to read. 25 plates takes approximately 50 minutes. Once Barrios scan is finished, remove the full magazine and place it on the magazine loading platform.
Lift up the outer rectangle of the platform and the magazine should slide off, leaving the plates standing in a pile in the middle. Finally, replace the lids on the plates for a solution-based screen. For cellulose activity first, prepare the assay mix using a pre-made 10 times stock of lysis.
Mix in 50 millimolar potassium acetate buffer at pH 5.5 as described in the written protocol. Then prepare a stock of up to 75 milligrams per milliliter, chromogenic DIPHENYL or DNP cell biocide substrate in DMSO, making sure the substrate is fully dissolved. Add DNP cell biocide stock solution to the assay mix to a final concentration of 0.1 milligrams per milliliter.
Keep in mind that each plate uses approximately 20 milliliters of solution and an additional 50 milliliters is necessary to allow for dead volume. Set up the Q fill three as before at the assay mix to each plate using the Q fill three. Each plate takes approximately 20 seconds to fill.
Once the assay mix is added, incubate the plates at 37 degrees Celsius for 12 to 16 hours. In a humidity box, remove the plates from the 37 degrees Celsius incubator. Set aside the lids and place the plates onto the magazine loading platform provided with the rapid stack in the same manner as before.
Open the scan its re program and set it up as done previously with the exception of the photometric measurement, which should be set to read at 400 nanometers. Then enter the parameters into the POLARA RS program. As was demonstrated earlier.
Allow the Vario scan to read the plates. Following the run, remove and dispose of the plates. To export the absorbance readings open, scan it re software and choose.
Open an existing file. Select the directory where the session was saved and hit the plus icon to expand the list. Under the title heading will be options labeled container one to container N.Depending on the number of plates, select the first plate to be analyzed in the menu bar at the top of the page, select data report export.
Choose options for export such as plate layout and photometric data. Click view report, then file save. Choose the desired directory.
The output format is a Microsoft Excel spreadsheet. Here, absorbance readings from a single 384. Well plate containing a positive clone as shown positive clones show a marked increase in absorbance as compared to clones not expressing cellulase activity.
Differences in assay time, well, location on the plate or DNP concentration can affect absolute absorbance readings. Relative absorbance readings such as the difference in absorbance above the plate average or column average are a more robust method of identifying cellulase positive clones. We've just shown you how to identify cellulase positive clones from a metagenomic library.
When doing this procedure, it's important to remember to dissolve your DNP cell bio side in DMSO and to incubate your plates in a humidity chamber. So that's it. Thanks for watching and good luck with your experiments.
