An Antibiotic Resistance Gene Using Recombinant DNA

Introduction

Recombinant DNA is the joining together of DNA molecules from two different molecules that are inserted into a host organism to make a new genetic combination. They are used in science,  medicine,  agriculture, and industry. Recombinant DNA was first brought out in 1980, it has helped a lot in the advancement of science. DNA recombination involves the process of cutting and joining DNA of different species through restriction enzymes and ligations (2).

The goal of this method is to have an antibiotic resistance gene using recombinant DNA. When it’s inserted into a host cell, it allows the bacteria to grow. The antibiotic gene is formulated from two relaxed plasmid reagents, pKAN and pUC19, each of which contains a single antibiotic resistance gene. These plasmids are digested in a pre-constructed hybrid restriction enzyme mixtures of BamHI and HindIII (3).

Each plasmid has a sequence where each enzyme cuts, making two restriction fragments which are produced and analyzed through a process called electrophoresis. Electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size through the passing of electric current (1). The cutting ability of hybrid restriction enzymes is confirmed and enzyme fragments are separated from the DNA fragments through electrophoresis.

On the other hand, ligation is a method that requires the presence of ATP and the ends of the fragments are fused by ligation (1). These ligations occur by hydrogen and phosphodiester bonds, which is what forms the recombinant DNA molecule. This recombinant DNA then goes through transformation with E. coli. Transformation is done in order to be able to select the antibiotic resistant gene. The colonies on the plates show how successful the transformation was and if the resistance was accomplished. X-Gal is used in the transformation process for identification purposes. It is a blue/white screening technique that allows for indication of whether a cell expresses a functional B-galactosidase enzyme in blue/white screening. The phenotype is then identified by conducting a purification procedure (4).

Materials and Methods

Materials

• pUC19 (0.20 ug/ul)
• pKAN (0.20 ug/ul)
• pUC19 (0.1 ug/ul)
• pKAN (0.1ug/ul)
• agarose gel
• BamHI/HindIII
• Ethidium bromide
• Loading dye
• Restriction buffer
• 1x TBE
• Beakers
• Electrophoresis supplies
• PPE equipment
• Micropipettor plus tips
• Tubes
• Water bath

Recombination of antibiotic resistance genes

Two plastic white centrifuge tubes were obtained and labeled; one pKAN and the other pUC19. Tubes were prepared according to table below.

After preparation, the tubes were incubated for 30 minutes at 37C. Once digestion was completed, the tubes were removed and spun down. 5ul of sample was removed from tube and placed in a fresh tube and 1ul of loading dye was added, and the tube was then placed in the freezer. This was done for both of the tubes. The digestion tube was put back into incubator for an additional 15 minutes. While waiting for that the gel was run and the 0.1ug/ul uncut plasmid was provided. When the 15 minutes digestion was complete, the tube was put into the digestion tub in 80C for 20 minutes to start the ligation process. Once the 20 minutes was complete, it was taken out and kept at room temperature for five minutes, then it was prepared according to the chart below.

Ligation:

The tube was incubated at room temperature for one hour then 4C for overnight incubation. Then the samples were stores at 0C until next class. For ligations, ATP is used because the energy given off of ATP is required. A general molecule of DNA is stable itself and doesn’t tend to bond with other DNA molecules: the 3′ end ends with a -OH group, while the 5′ end ends with a phosphate group. The DNA ligase then removes the OH^– and binds the two segments together.

Materials

• CaCl2 (50mM)
• Mid-log MM294 cells (two 10ml cultures)
• Beakers for ice
• Bunsen burner
• Centrifuge
• Micropipettor
• Pipettes
• Water bath
• Shaking incubator
• Sterile loop

Transformation:

While performing this exercise it was important to make sure everything was on ice throughout the experiment. Three plastic tubes were labeled the following way; +lig, +pUC19, and +pKAN. The cells were added to each of the tubes, then the pDNA was added to the tubes and placed on ice. All tubes should stayed on ice for 15 minutes. The cells were carried in a beaker of ice to the 42C where cells were heat shocked for 90 seconds. Following that, they were returned to ice for one minute. Next, 500uL of LB broth was added to the tubes with the p1000 pipettes. And then the tubes were ready for recovery, which would be in the shaking incubator. The tubes were put into the shaking incubator at 37C for about 60 minutes. In the meantime, three plates of each type were obtained and labelled: LB/Amp/IPTG/XGal, LB/Kan/IPTG/XGal, and LB/Amp+Kan/IPTG/XGal.. Set “A” plates were for ligation and should be labeled with the letter set “B” were for pUC19 and set “C” is for pKAN. Set A was labelled with “L”, B with “A” and C with “K.” Once the 60 minutes were done, it was time to plate. 100ul of each tube were plated on each plate. One plate was done at a time and the hockey stick was flamed between each spread to avoid contamination. The tubes were then incubated for 24 hours.

Materials

• Agarose gel supplies
• BamHI/HindIII
• Ethidium bromide
• Lambda DNA
• Loading dye
• Miniprep DNA/TE
• pUC19 (0.1 ug/ul)
• pUC19/pKAN (0.1 ug/ul)
• restriction buffer/RNase
• 1x TBE
• Beakers
• Pipettors
• Tubes
• Water baths
• Electrophoresis supplies

Isolation of plasmid DNA:

Two cultures were obtained, both of which consisted of E.coli MM294 containing a plasmid. Two microcentrifuge tubes were labeled with identification. A 1.5mL of cells was spun down in each of the microcentrifuge tubes for two minutes. Once the two minutes were up, the supernatant was poured off into a waste beaker containing bleach and then the process was again with the remainder of the sample.

Plasmid preparation:

100uL of cold GTE was added to the sample. The pellets were resuspended using the vortex mixer or a pipette. After, resuspension, add 200uL of SDS/NaOH buffer was added, and rapidly mixed by inversion. Once mixed, the tube was set aside for 5 minutes on ice. The suspension was clear at this point. Next, 150uL of cold KOAc was added and mixed by inversion, which caused the protein and cell membranes to precipitate. After mixing, the tubes were placed on ice for 5 minutes again then spun to remove the precipitate. Next, 400uL of the suspension was transferred to a new tube. The suspension was mixed rapidly with an equal volume of isopropanol, then it was left at room temperature for 5 minutes. After 5 minutes, the tubes were spun down for an additional 5 minutes to pellet the pDNA. Next, the supernatant was taken out. The procedure was then repeated with 200uL of 100% ethanol to clean the DNA of any residual salt. Once DNA was cleaned, the tube was dried with direct air from a heat gun. After all ethanol evaporated and the pellet was completely dry, the pellet was resuspended in 15ul of TE.

Restriction Digest: 

The chart shown below was used to set up the second digest. Each tube had the following samples in it.

Once the tubes were prepared, they were incubated at 37C for 45 minutes. In the meantime a well agarose gel was prepared. Once incubation was completed, the gel was loading as follows:

M1-, M2-, A-, K-, lambda ladder, M1+, M2+, AK+

Results

 Figure 1: pKAN and pUC19 Digestion

Figure 1 shows the first digestion of pKAN and pUC19.

Table 1: Transformation

Table 1 shows the results from the plates and how much grew on each plate. The results weren’t as expected.

 Figure 2: Miniprep digestion

Figure 2 shows the miniprep digestion. Lane 1 has miniprep 1 uncut, Lane 2 has miniprep 2 uncut, Lane 3 has A uncut, Lane 4 has K uncut, Lane 5 has lambda ladder, Lane 6 has miniprep 1 cut, Lane 7 has miniprep 2 cut and lane 8 has AK cut.

Transformation Efficiency

DNA in cell sample = Concentration of plasmid X volume of plasmid

(0.2 ug/ul) x (24 ul) = 4.8 ug

DNA on plate = DNA in cell sample  X  volume of cells spread on plate

                       Total volume of cell sample

(4.8 ug) x (100 ul) /

Transformation Efficiency = number of colonies

                  DNA on plate                              

Discussion

 We successfully completed all the digests and ligations in the laboratory but we could not achieve the formation of a recombinant DNA molecule. The first digest worked and cut the enzymes as we hoped it would. Although our ligation was done properly, the transformation didn’t work. The plates had no growth which means it was unsuccessful. This could have happened because of a few reasons; the digest of pKAN and pUC19 didn’t yield enough DNA to finish the ligation, the tubes had to be in the heat shock for longer than 90 seconds, or maybe the ligation had to be run for longer. Whichever caused the transformation to be unsuccessful inhibited the DNA from ligating, however, there are future precautions that can be taken to make sure this can be avoided. Properly and accurately digesting DNA is important. It is easy to make a mistake when making the samples and following the little steps so paying close attention is a good start. Also, it wouldn’t be harmful to extend the time for the heat shock and the ligation time by just a little.

 Even though our results didn’t turn out as expected, we can still analyze what was supposed to have happened if the experiment was successful. Some results include; the A LB/amp plate should have had more growth than the L LB/amp plate due to the mass of the ligated DNA. The A LB/kan plate shouldn’t have had any growth because of the resistance gene present in it. The L LB/kan plate should have had growth compared to the A LB/kan plate because of the presence of kanamycin. There should be growth on both, the A LB/amp and K LB/amp plates, but the A LB/amp should have a little bit more because of the ampicillin. The L LB/amp should have more growth than the L LB/kan because of the kanamycin. The L LB/amp should have more growth when compared to the L LB/amp+kan. And lastly, the due to the antibiotic present in both of these, L LB/kan and L LB/amp+kan had the same amount of growth.

 During our second digest we completed the experiment according to what was given. When we ran the gel after the digest, the uncuts showed but not the cuts. This could have happened for a few different reasons. It is possible that the digest wasn’t run properly, something that needed to be in the tubes could have been missed. Also, it’s possible not enough sample was loaded into the gel was caused it to not show. It is also possible that not enough loading dye was added to the samples before loading the gel. It is always important to carefully read each step before preforming the exercise as it is easy to miss the little steps, however, future precautions can be taken to avoid this from happening again. Simply by reading everything thoroughly, a lot of small and simple errors can be eliminated. Making sure you are adding the right amounts of all the samples is also very important. You can also have the digest run for a little longer to make sure everything does cut.

Literature cited

1. Freeman WH. Gel Electrophoresis. Nature News. 2014. https://www.nature.com/scitable/definition/gel-electrophoresis-286
2. “Genomic Determination of the CR1 (CD35) Density Polymorphism on Erythrocytes Using Polymerase Chain Reaction Amplification and HindIII Restriction Enzyme Digestion.”Journal of Immunological Methods, Elsevier, 12 Nov. 2002, www.sciencedirect.com/science/article/pii/0022175991900062
3. Griffiths AJF. Recombinant DNA technology. 2018 Nov . https://www.britannica.com/science/recombinant-DNA-technology
4. Micklos, D. A, Freyer, G. A, et al. DNA Science a First Course. 2nd ed., Cold Spring Harbor Laboratory Press, 2003.