Sequencing reference genomes for various built environments requires a series of steps. The first part of the procedure is to sample an environment that possibly has interesting microbes. For an example one might sample a toilet, public tables, floors or human associated items. There are 2 ways to sample something, direct plating or indirect plating. Direct plating is done by directly swabbing the area of interest with a sterile swab then directly swabbing onto a plate. Indirect plating is done by swabbing the area then putting the swab directly into media and allowing growth to occur, then plating.
The next step after growing the samples is to dilution streak. Dilution streaking is a method used to isolate a single organism by systematically reducing the amount of bacteria in different parts of the nutrient plate. To dilution streak, first sterilize an inoculation loop and run it through a colony in the sample plate. Take the inoculation loop and create a heavy “pool” of microbes in one corner of the new plate. After doing this, sterilize the inoculation loop again and drag it through the pool creating a zig zag pattern. Repeat this step one more time using the previous pattern as the “pool”. This allows individual colonies to form.
After the individual colonies have grown, it is time to make over night cultures. To do this take 5ml of the appropriate media and with the sterilized inoculation loop, scrap a colony and put it into the liquid. After the overnight culture as grown we can extract genomic DNA by doing genomic preparations. Genomic preparation involves lysing the membrane and nuclei, which allows the DNA to flow into the solution. Cellular proteins and other impurities are then removed using salt-precipitating solution and isopropanol. Lastly, the genomic DNA is suspended in rehydration solution for further analysis. After this, we must confirm there is actually genomic DNA. To do this, we use an agarose gel. A mold of agarose solution is created with wells. The mold is placed in a “gel box” with TAE buffer. The genomic DNA and a UV-fluorescing dye are loaded into the wells and a current of 120 V is run through it for 20 minutes. This separates the DNA fragments. We then analyze the DNA fragments under a UV light. If DNA fragments are present, that means there is genomic DNA.
The next step is to do a 16S PCR. A PCR is involves ribosomal RNA which is highly conserved, and is done to amplify this ribosomal RNA. This procedure involves a master mix made up of Taq Buffer, Q Buffer, dNTP, P1 P2 Primers, Taq polymerase, water and DNA. Then we must confirm the PCR using agarose gel electrophoresis. Always have a positive and negative control and ladders. The gel allows us to see if the PCR was correctly done with a presence of a band at 1300bp and by looking at the size of the bands.
After 16S PCR is confirmed the PCR is cleaned up. This step requires Buffer NT-Bind 16S DNA to membrane, NT3 buffer-wash PCR reagents, dry silica membrane, and Elution- Buffer NE. The cleaned PCR is then TOPO cloned. To TOPO clone, use the Invitrogen protocol and kit. Take a few micro liters of PCR product and mix with TOPO vector, the enzyme ligates the PCR product to the vector. Then the plasmid is transformed into E.Coli by mixing the TOPO reaction with E.Coli and incubating on ice. The cells are then heat shocked to allows plasmids to enter. Then the cells are allowed to repair so ampicillian resistance can be expressed. Spread the transformation onto LB carbenicillian plates and incubate overnight. Plasmid preps are then made by first picking the correct colony and grow in an over night culture. Then the plasmids are isolated from the E.Coli following the Qiagen kit protocol. Various buffers are added to break down cell walls and precipitate protein then transfer into QIAprep spin column to capture plasmid DNA. Add more buffers to isolate plasmid DNA from genomic DNA then elute plasmid DNA into clean microcentrifuge tube by washing out the spin column.
The plasmid is then quantified using plasmid quantification procedures. Use a spectrophotometer to measure how much DNA was isolated during the plasmid prep. This shows us the amount of DNA that is in the sample, this is important because a certain amount of DNA is needed before the sample can be sent to be sequenced. Use the dsDNA function and mix 1 micro liter of sample with 99 micro liters of water. Multiply the reading by 100 to take into account the dilution factor. After getting the readings the plasmids and/or the PCR is diluted to specific concentrations and transferred to the UC Davis DNA sequence center. A few days later the results are sent back to us. Using the Geneious program we edit and analyze the gene sequence. We copy and past the cleaned up sequence onto Blast website to compare the sequence of our 16s to other organism. Lastly we use Genome online database (GOLD) to see genome sequencing projects relating to our organism. We check the complete, incomplete and targeted projects to see what has been done thus far. From here we decide whether or not to go further into research.