Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

The overall goal of the following experiment is to produce TROs of a wild type bacterial strain that grow in minimal salts medium supplemented with glucose. This is achieved by transforming a transpo zone into the wild type host strain to obtain a canna mycin resistant population of bacteria with random transpose on inserts. Next transformants are screened by replica plating, which identifies TROs that grow on rich medium, but fail to grow on minimal salts, medium supplemented with glucose.

Next genomic, DNA from the mutant is digested ligated and transformed into e coli in order to obtain a plasmid containing the transposon flanked on each end by segments of the interrupted gene. Results are obtained that show the putative identity of the interrupted gene based on DNA sequencing and basic local alignment search tool analysis. The transposon used in this technique has a replication origin, a kanamycin resistant gene, and mosaic ends.

For transposes protein binding. It lacks transposes protein gene and hence we have to add this transposes gene with the transposon DNA fragment, and then the complex between the transpos DNA and the protein is electroporated into the host. The main advantages of electroporation are the elimination of the need for transduction or conjugation.

The lack of a transpose a gene usually produces stable inserts and the presence of a replication origin and a CIN resistance gene allow for easy identification of the mutated gene, which is recovered as a recombinant plasmid. This method can help answer key questions in the microbial physiology field, such as identifying genes required for the biosynthesis of amino acids, nucleic acids, and vitamins. I first had the idea for this method when I was searching for metal resistance genes in enterobacter species YSU and decided to incorporate it into a microbial physiology lab that I was developing To electroporated competent cells using an overnight culture of enterobacter species.

YSU prepare a one to 20 dilution in fresh LB medium and incubate at 30 degrees Celsius and 120 RPM to an OD 600 between 0.4 and 0.6. When the cells have reached the proper density, chill them on ice for five minutes and then centrifuge at four degrees Celsius and 7, 000 Gs for five minutes. Discard the supernatant and use an equal volume of ice cold water to Resus.

Suspend the cells centrifuge at four degrees Celsius and 7, 000 Gs for five minutes. After repeating the wash, discard the supernatant and with ice cold water resus suspend the cells in a volume equal to the volume of the cell pellet. Next, add 0.5 microliters of the transpo zone to 40 microliters of cells.

Then place the cell transpo zone mixture in an ice cold electroporation vet and tap the mixture to the bottom of the Q Vet shock the cells at 25 micro phs, 200 ohms and 2.5 kilovolts. Immediately add 960 microliters of filter sterilized SOC medium mixed by pipetting up and down before transferring the cells to a sterile 1.5 milliliter micro fuge tube. Incubate the cells at 30 degrees Celsius and 120 RPM for 45 to 60 minutes.

Then spread 100 microliters of cells on an LB can agar plate before incubating the plate overnight at 30 degrees Celsius to grid Transformants. Tape a grid to the lid of an empty 100 by 15 millimeter Petri dish. Draw a line on the bottom of an LB can agar plate and use a small piece of tape to anchor it to the gridded lid so that the grid is visible through the auger With a sterile toothpick.

Choose a single transformant and spot it in the center of a square in the grid spot another transformant in an adjacent square and continue until the plate is full. Then incubate it overnight at 30 degrees Celsius. This is the master plate for each plate that was grided.

Make a stack of three fresh auger plates numbered one through three. Number one for LB can number two for M nine can. And number three for LB can dry the plates upside down overnight at 37 degrees Celsius before use.

After drawing a line on the top of each plate as demonstrated earlier, use 70%ethanol to wipe the replica plating tool and place a sterile veltin square on top of it before clamping the veltin square down. Next, align the line on the master plate with a line drawn on the clamp and place the plate on top of the veltin square. Apply gentle pressure to transfer the bacteria from the plate to the veltin square.

Then line up the line drawn on plate number one with the line on the clamp and place it on top of the veltin square. Apply gentle pressure to transfer the bacteria from the veltin square to the plate. Remove plate number one.

Discard the velvet teen square into a beaker of water and place a clean sterile velvet square on the replica plating tool. As before, line up the line on plate number one with the line on the clamp and place it on top of the new velvet square. Apply gentle pressure to transfer the bacteria from plate number one to the velvet square.

Remove plate number one, line up the line drawn on plate number two with a line on the clamp and place it on top of the velvet square. Apply gentle pressure to transfer the bacteria from the velvet teen square to plate Number two, remove plate number two after repeating the process for plate number three, incubate the plates overnight at 30 degrees Celsius. Colonies that grow on both LB can plates but fail to grow on M nine plates are considered troves.

Choose TROs from plate number one and streak onto a fresh LB can plate. After incubating overnight, choose three colonies from the streak plate and streak them out onto a fresh LB can plate the next day. Spot colonies from the newly streaked plate onto an LB can grid plate and use this plate to repeat the replica plating as just demonstrated.

Carry out gene rescue. Use an isolated mutant colony to grow a three milliliter lb. Can overnight culture at 30 degrees Celsius.

Then use a commercially available genomic DNA purification kit to purify DNA from one milliliter of the culture at 0.025 units of BFUC one restriction endonuclease to 14 microliters of one microgram per microliter of genomic DNA in a 20 microliter reaction on ice. Then incubate the reaction at 37 degrees Celsius for 25 minutes before transferring it to 80 degrees Celsius for 20 minutes. To inactivate the enzyme, analyze three microliters of the partially digested DNA on a 0.8%AROS gel.

Next, add 800 units of T four DNA ligase to 15 microliters of partially digested genomic DNA in a 500 microliter reaction and incubate the reaction at four degrees Celsius overnight the following day. Precipitate the ligated DNA by adding 50 microliters of three molar sodium acetate pH 5.5 and one milliliter of 95%Ethanol incubate at negative 20 degrees Celsius for 20 minutes. Micro fuge the DNA at four degrees Celsius and 13, 500 Gs for 10 minutes.

Pour off the supernat venues, seven 70%ethanol to wash the pellet. Dry it in a centrifugal vacuum concentrator and use 10 microliters of nuclease free water. To resuspend the DNA Use four microliters of resuspended DNA to transform by electroporation e coli strain, ECD 100 DPIR or ECD 100 DPIR one 16 inoculate five milliliters of LB can medium with a single colony.

Grow it at 37 degrees Celsius and 120 RPM overnight. The next day after purifying the DNA from the entire culture, use Joe one to digest 14 microliters of the plasmid. Analyze 10 microliters of the undigested and digested plasmid on a 0.8%agarro gel.

After sequencing the plasmid search for the sequence five prime GA, G-A-C-A-G three prime, which is the last seven base pairs of the transpose on and will be followed by the sequence for the interrupted gene. Use blast to determine the possible function of the interrupted gene. A successful electroporation yielded several thousand transformants each with at least one transpose on insert in this figure.

The black squares show two colonies that grew on the LB can plate, but not on the M nine minimal medium plate. These TROs probably contained interruptions in genes involved in the synthesis of an amino acid, a nucleic acid, or a vitamin, which are all found in lb, but not in M nine, minimal medium. The other 86 colonies did not have an interruption in a gene that is required for growth on M nine.

Minimal medium as seen here, partially digested genomic DNA from an isolated TRO runs from above 10 kilobases to below 0.5 kilobases in size. The rescued plasmid consists of the two kilobase transposon plus flanking host DNA. This figure shows a blast result for a plasmid that was isolated from an tro.

The interrupted sequence is similar to an enterobacter cloy gene for ate mutase, which is involved in the biosynthesis of L tyrosine, L phenylalanine, and L tryptophan Due to multiple overnight growth steps. This technique can be completed in 10 or 11 days if performed properly. While replica pla, it's important to remember not to smear the plates, and you can prevent that by using dry plates and by not moving the plates once it has been placed on the veltin square.

Following this procedure, other methods like cloning or complementation experiments can be performed to verify the function of the identified gene. After watching this video, you should have a good understanding of how to generate TROs using transposon mutagenesis, and how to tentatively identify the mutated genes.