Microbial Culture

 Introduction  Again, in this series,  Deinococcus radiodurans  and  E. coli  are amplified to isolate certain genes and block a pathway of  D. radiodurans .  E. coli  is used for its KanR gene, which is responsible for the bacterium's ability to resist the antibiotic kanamycin.  The KanR gene in this assay is used to block a specific gene within  D. radiodurans  known as ZWF of the pentose phosphate pathway. The blockage of this gene will present opportunities for future studies of  D. radiodurans  in its behavior and ability to survive under extreme conditions. The insertion of the KanR gene is important because it will be used as an indicator that the DNA has been successfully integrated into the DNA sequence due to  D. radiodurans  becoming resistant to kanamycin.  Procedures  The  E.coli  KanR plasmid was extracted using a Monarch Plasmid Miniprep Kit. The pressure for this bacteria ...

Introduction  This is a continuation of the previous lab report. The fragments of Deinococcus radiodurans were amplified via PCR and were verified through Gel Electrophoresis. The right fragment was successfully extracted from the gel and cleaned of debris. The left fragment did not show which indicated a failure in the procedure of that fragment. The left fragment had the contents of Tube 1, so there may have been a miscalculation of concentration.  The next step will be to correct this error, the volume measurements from Tube 2 will be used to remake the left fragment to correct this issue.  Procedures  Deinococcus radiodurans  DNA was extracted using a DNeasy Ultra Clean Microbial Kit.  Then, the extracted DNA was prepped for PCR.   A total volume of 20 uL was used, the previous reaction included: 17 uL of a Q5 master mix, 1 uL of each primer (totaling 2 uL) and 1 uL of the genomic DNA; This round included: 14 uL of a Q5 master mix, 2 uL of ea...

 Introduction  PCR is an abbreviation for Polymerase chain reaction, a laboratory technique used to multiply a specific segment of DNA.  PCR uses short synthetic DNA fragments called primers to isolate a specific segment of a gene to be amplified.  Then multiple rounds of DNA synthesis are conducted to amplify that segment, producing  millions of copies of the specified gene. In this series, Deinococcus radiodurans  and E. coli are amplified to isolate certain genes and block a pathway of D. radiodurans . E. coli is used for its KanR gene, which is responsible for the bacterium's ability to resist the antibiotic kanamycin.  The KanR gene in this assay is used to block a specific gene within D. radiodurans known as ZWF of the pentose phosphate pathway. The blockage of this gene will present opportunities for future studies of D. radiodurans in its behavior and ability to survive under extreme conditions. The insertion of the KanR gene is important be...

What is Gibson Assembly?   Gibson Assembly is a technique used to insert a single fragment or multiple fragments into a vector.  A vector or genetic vector is an instrument used as a delivery method to transmit foreign DNA to recipient cells.  The vector that will be used is the E. coli plasmid due to a particular interest in its kanamycin resistance gene.  Gibson Assembly allows gene transformation without the use of restriction enzymes.  Restriction enzymes are used as a defensive mechanism in bacteria against bacteriophages and can be used to manipulate genes within DNA.   What are restriction enzymes?  Restriction enzymes can be disruptive in gene manipulation.  These structures are meant to keep foreign entities out of the cell so it is possible that the restriction enzymes may cut in many sections of the gene causing a lack of overlap which would decrease the chances of ligation of the gene that is desired for insertion.  Wha...

 Introduction Previously, it was found that the culture had been contaminated by a yellow bacteria that outgrew the Deinococcus radiodurans . It was suspected that the contamination came from an outside source, like the air from not keeping the inoculation loop properly under the flame.  Other suspicions are that the tube was used and not properly cleaned so the bacteria invaded the culture from within.  To determine if the culture broth, within the tube, was contaminated or if it was just the agar plate that was contaminated, a new agar dish was prepared with the cultured broth and incubated for 5 days.  Materials & Procedure The materials that were used for t his anal ysis were as follows: a disposable inoculation loop, to reduce the chance of contamination, a P200 pipette, 50 μL of the cultured broth that is suspected of contamination, a new agar plate, and an incubator set at 27 °  celsius.  First, with the agar plate ready for streaking, take the P...

 Introduction  Previously, a hypothesis was tested that if a previously incubated 15mL tube with a tight lid was re-incubated, the bacterial cells would grow.  This test came out to be true, in this instance. Those bacterial cells were used to grow colonies on an agar plate.  Material & Procedures To culture the agar plate, the following items are needed: an agar plate, cultured liquid media (broth), a metal inoculation loop, a bunsen burner, a striker, and an incubator.  First, install the bunsen burner to a gas source.  Once it is installed, turn on the gas, there should be a low hissing sound when the gas is released, and use the striker to ignite the flame. Now, take the inoculation loop and sterilize it using the flame allowing it to burn until it is glowing red, sterilize all parts that will enter into the container that contains the cultured broth.  Once it's ready, allow the loop to cool down, being mindful to keep it under the flame (next ...

Introduction Previously, a 15mL tube with 5mL of TGY broth with live cultures of D. radiodurans was incubated for 48 hours. When the incubation process was stopped, there was little to no growth resulting in an OD600 reading of -0.06 nm.  There was a small orange-colored pellet at the bottom of the tube and the media was clear with a slight orangish tint.  This led to a hypothesis that the lack of growth could have been from the lack of oxygen allowed to flow into the tube, hindering the growth process. So, if more oxygen was allowed to flow into the tube then there will be more growth of the cells.  Materials & Procedure Materials that were used are as follows: A cultured TGY broth with  D. radiodurans cells, from part 1 of this experiment in a 15 mL centrifuge tube, a large tub, balance tubes, and an orbital floor shaker.  A vortexer, P20 pipette, kimwipes, deionized (D.I.) water, uncultured TGY broth, and a nanodrop (nanometer) were also used in the resu...