Mutagenesis in pGLO Bacteria 1 Mutagenesis in pGLO Bacteria Alissa Eckas and Elizabeth Jorgensen University of Northern Colorado Mutagenesis in pGLO Bacteria 2 Abstract In a society where antibiotic resistant super bugs are becoming more and more prevelant it is important to determine where in the bacterial genome the bacteria is susceptible to antibiotics and in what circumstances the bacteria will fail to thrive. By using E. coli bacteria and a Tn5 transposon we were able to test where in the three genetic coding regions the transposon inserted and mutated. A green colony indicated that the araC gene bound to the GFP promoter fed by the arabinose sugar present. As a control a plate utilizing only LB broth and no DNA was plated, in the absence of sugar and E. coli DNA only white colonies were possible as arabinose is required for the green florescent protein to activate. Further, as it is mutagenized bacteria that is able to grow in the presence of ampicillin or kanamycin we needed a control to show that bacteria was in fact present in the samples and capable of producing colonies. Introduction Bacterial plasmids have been utilized in genetics research for a broad range of applications including: functional DNA analysis, gene targeted therapies for applications such as vaccines and new antibiotics, DNA sequencing and identification of proteins (Choi & Kim, 2009). Plasmids serve many purposes within the bacterial genome, including extending antibiotic resistance to new cells and promotion of bacterial mating thus creating exponentally more copies of the bacteria being studied (Pierce, 2014). The pGLO plasmid being utilized for this experiment contains three distinct genes that affect the growth of the E. coli bacteria depending on the insertion site of the transposon. A green florescent protein (GFP) has been inserted into the pGLO plasmid, in the presence of this protein bacterial colonies will glow green under UV lights (Leatherman, 2018). Also present is a gene that encodes for the AmpR enzyme. We are able to test for the presence of this enzyme by plating samples on ampicillin containing Mutagenesis in pGLO Bacteria 3 plates and observing growth or lack thereof (Leatherman, 2018). Further for the E. coli bacteria to grow in the presence of ampicillin the protein beta-lactamase is needed as it acts to inactivate ampicillin (Leatherman, 2018). The araC gene acts as a regulator of the GFP gene, araC binds to the promoter in GFP transcription, when arabinose is present araC binds to the promoter leading to glowing green colonies (Leatherman, 2018). The Tn5 transposon was used to mutagenize the pGLO plasmid in our study. A transposon is a short piece of DNA that randomly inserts into the plasmid and can render the gene non-functional. Transposons are also referred to as “jumping genes” because of their ability to shift from position to position in the genome causing chromosomal mutations along the way (Choi & Kim, 2009). The insertion point of the Tn5 transposon in our pGLO plasmids will be determined by use of electrophoresis testing. Digest enzymes are used to “cut” the DNA and predict where the transposon inserted into the genome. By using different restriction enzymes we are able to determine if the transposon inserted in a region expected based on the plating results. We expect the transposon regions to appear longer and darker in the mutagenized bacteria. Methods Mutagenesis To facilitate insertion of the Tn5 transposon into the pGLO plasmid, pGLO plasmids were combined with deionized water, EZ Tn5 10X reaction buffer, 0.1 p mol/microliter Tn5 transposon and EZ-Tn5 transposase. The solution was centrifuged to incorporate all components. The solution then underwent a series of incubations at 37oC, was recentrifuged and a stop solution was added. Finally, the solution was heat shocked at 70oC and then stored in a freezer. Mutagenesis in pGLO Bacteria 4 Transformation Chilled calcium chloride solution was added to three tubes of chilled and centrifuged E. Coli cultures. A series of 4 cooling and centrifugation cycles were completed prior to the addition of DNA. Tube one was the control tube with no DNA added. Wild type pGLO DNA solution was added to tube two. Tn5 mutagenized pGLO DNA was added to tube 3. The tubes were then incubated on ice prior to being cooled, heat shocked, cooled again and then incubated at 37oC for 30 minutes while being shook. Six plates were prepared: lysogeny broth (LB) without DNA, no DNA and ampicillin, wild type pGLO DNA and ampicillin, wild type pGLO DNA with ampicillin and arabinose, mutant pGLO DNA with kanamycin and arabinose and finally wild type pGLO DNA with kanamycin and arabinose. The plates were incubated overnight at 37oC, after incubation the plates were stored in a refrigerator. Purification Two sterile culture tubes were prepared for this step, one with LB and ampicillin, the second with LB and kanamycin. A wild-type pGLO colony was taken from the arabinose plate and pipetted into the LB and ampicillin solution. A mutant pGLO colony was taken from the kanamycin and ampicillin plate and pipetted into the LB and kanamycin solution. The cultures were incubated in a shaker incubator overnight. A sterile loop was used to obtain a sample from the kanamycin resistant solution, the loop was then streaked over an ampicillin and arabinose plate and incubated overnight. The bacterial cultures were centrifuged to separate the bacterial pellet from the supernatant, the supernatant was discarded. Buffer solutions P1, P2 and N3 were added to the pellet in order and pipetted up and down to mix. The solutions were then centrifuged twice more, the second time in a spin column to allow for the collection of DNA supernatant. Buffer Mutagenesis in pGLO Bacteria 5 PE was then added to the DNA supernatant. The DNA was then centrifuged twice more to remove any wash buffer. Fifty microliters of deionized water was then added to the DNA supernatant before centrifuging. The DNA is now located in the spin through portion of the tube. DNA concentration readings were taken using a Nanodrop. The wild type concentration was 111.2 ng/ul, the mutagenized concentration was 176.5 ng/ul. DNA Restriction Digests and Gel Electrophoresis Restriction enzymes Nde I, Nae I, Xmn I, Xae I and Xmn I were used to determine where the transposon inserted into the bacterial plasmid. Three digest tests were set up and applied to both the wild type and mutant plasmids. Digest 1 consisted of Nde I and Nae I, digest 2 consisted of Xmn I and Xae I, finally the third digest consisted of Nae I and Xmn I. The restriction digests were composed of DNA, 10X buffer, the restriction enzyme and deionized water. The amount of water, enzymes, and 10X buffer were calculated based on the DNA concentration of the wild type and mutant pGLO. The restriction digests were incubated at 37oC for 1.5 hours, then the tubes were removed and stored in the freezer. Agarose gel was prepared using 1X Tae buffer with agarose powder. The agarose solution was heated to dissolve the gel granules in the buffer and then allowed to cool before pouring the mixture into a prepared agarose horizontal gel apparatus. The gel was then covered with 1X Tae buffer. The restriction enzymes were prepared by adding 10X loading dye to each tube. The restriction enzymes were pipetted into the slots left from removing the comb. A 10uL 1kb ladder molecular weight marker was used as a control. The gel electrophoresis ran until the dye could be seen halfway down the gel. The gel was then removed, placed in a staining box and just covered with SYBR green staining mix. The staining box was covered in aluminum foil to prevent light from affecting the dye reaction. Mutagenesis in pGLO Bacteria 6 Results To test where the Tn5 transposon inserted into the E. coli bacteria DNA six experimental plates were set-up as shown in Table 1. The use of plates containing ampicillin without arabinose sugar, plates with both arabinose and arabinose and finally kanamycin and arabinose allowed us to predict colonial growth. The results of the plates were not all consistent with what was expected. Table 2 shows the outcome of our experiment. In the case of the wild type pGLO with ampicillin we expected to see white colonies but no colonies were present. This may have been the result of contamination of the plate when preparing the plate or failing to store the plates upside down during the initial growth period. Additionally, in the wild type pGLO with ampicillin and arabinose plate we expected to see a green colony because the plasmid inserted was expected to be ampicillin resistant and the GF protein flourishes in the presence of arabinose causing the typical green glow. Again the most likely cause of this outcome was contamination during the initial plating and possibly failing to store the plates upside down. The remaining four plates had outcomes as expected. The mutant pGLO with kanamycin and arabinose plate produced few green colonies but colonies were seen. The LB and no DNA and no antibiotics plate performed the best with a significant coating of white colonies observed. We chose to take colonies from the mutant pGLO with kanamycin and arabinose plate for the later stages of our experiment because this plate both contained DNA and showed the expected results. In preperation for electrophoresis gel testing the DNA samples had to be combined with deionized water, 10X buffer and the restriction enzymes being used. Tables 3 and 4 show the specific amounts of each element used to form the eight test samples. The level of 10X buffer and enzymes were given. The amount of DNA needed was determined by the formula C1V1=C2V2 where we have a known final concentration 0.5ug, and a known intitial Mutagenesis in pGLO Bacteria 7 concentration per volume ug/uL. The amount of water needed was determined by subtracting the known amounts from 20uL to react a final volume of 20 uL. Table 5 shows where we expect the digest restriction enzymes to cut the DNA. Digest one contained Nde I and Nae I and was expected to cut the DNA at position 1612 for AmpR, and position 3312 for the non-coding regions between GFP and AmpR and ORI and araC respectively. Digest two contained Xmn I and Xae I and is expected to cut AmpR at position 5851, cut the non-coding region between GFP and AmpR at position 1286 and finally to cut the non-coding region between ORI and araC at position 5851. Digest three contained Nae I and Xmn I, this digest is expected to cut AmpR at position 2874, the non-coding region between GFP and AmpR and the non-coding region between ORI and araC at position 2807. Figure one shows the results of our gel electrophoresis tests. The results seem to indicate that in our first mutant test that the transposon inserted at around position 3,000 which indicates that the transposon was inserted in the non-coding region between ORI and araC. Our second mutant digest seems to have inserted around position 6,000 meaning that we expect that this transposon inserted in the AmpR coding region. Finally our third digest has inconclussive results, the extended streak of white on the gel seems to suggest that the transposon inserted between position 5,000 and position 2,000 as we expected this digest to show a change around position 2,800 it is my conclusion that this digest does not show a valid result but rather may have been contaminated or the concentration calculations weren’t correct. Figure two is the genomic mapping of the pGLO plasmid. The blue lines indicate the the cut sites of the Nde I restriction enzyme at positions 1340, 1574 and 4927. The purple lines show where the Nae I restriction enzymes cut at positions 3365 and 5318. The green line shows where we expect the Xma I resrict enzyme to cut at position 2079. Finally the orange lines show Mutagenesis in pGLO Bacteria 8 where we expect the Xmn I enzyme to cut at positions 2144 and 2823. DNA Added (no DNA, wt, mutant) LB and no DNA No DNA Wt pGLO Wt pGLO Wt pGLO Mutant pGLO Type of Plate No antibiotics Ampicillin Ampicillin Ampicillin and arabinose Kanamycin and arabinose Kanamycin and arabinose Prediction White colony No colony White colony Green colony No colony White or green colony Table 1: Experimental Plate set-up DNA Added (no DNA, wt, mutant) LB and no DNA No DNA Wt pGLO Wt pGLO Wt pGLO Mutant pGLO Type of Plate No antibiotics Ampicillin Ampicillin Ampicillin and arabinose Kanamycin and arabinose Kanamycin and arabinose Outcome White colony No colony No colony No colony No colony Green colony Table 2: Experimental Plate Results Wild-Type pGLO Water 10X Buffer DNA: 0.5 ug (ug/uL of stock 111.2) Enzyme 1 (20 units/ uL) Enzyme 2 (20 units/uL) Uncut 13.51 uL 2 uL 4.49 uL None None Digest 1 11.51 uL 2 uL 4.49 uL 1 uL 1 uL Digest 2 11.51 uL 2 uL 4.49 uL 1 uL 1 uL Digest 3 11.51 uL 2 uL 4.49 uL 1 uL 1 uL Digest 2 13.17 uL 2 uL 2.83 uL 1 uL 1 uL Digest 3 13.17 uL 2 uL 2.83 uL 1 uL 1 uL Table 3: Wild-Type pGLO Digest Rescription Enzyme Solution Preperation Mutant pGLO Water 10X Buffer DNA: 0.5 ug (ug/uL of stock 176.5) Enzyme 1 (20 units/ uL) Enzyme 2 (20 units/uL) Uncut 15.17 uL 2 uL 2.83 uL None None Digest 1 13.17 uL 2 uL 2.83 uL 1 uL 1 uL Table 4: Mutant pGLO Digest Rescription Enzyme Solution Preperation Wild Type pGLO AmpR Non-coding region between GFP and AmpR Non-coding region between ORI and araC Uncut 5371 5371 & 1221 5372 & 1221 5373 & 1221 As Expected Yes Yes No No Yes Yes Mutagenesis in pGLO Bacteria 9 Wild Type pGLO AmpR Non-coding region between GFP and AmpR Non-coding region between ORI and araC Digest #1 1393, 234, 2091, 1262, 391 1393, 234, 2091, 1262, 1612 282, 234, 3312,1262,391 1393, 234, 3312, 1262, 391 Wild Type pGLO AmpR Non-coding region between GFP and AmpR Non-coding region between ORI and araC Digest #2 65, 679, 4630 65, 79, 5851 1286, 679, 4630 65, 479, 5851 Wild Type pGLO AmpR Non-coding region between GFP and AmpR Non-coding region between ORI and araC Digest #3 2132, 1586, 1653 2132, 1586, 2874 2132, 2807, 1653 2132, 2807, 2874 Table 5: Expected Transposon Insertion Sites of Digests Uncut Mutant Uncut Wild Type Digest 1 Mutant Digest 1 Wild Type Digest 2 Mutant Digest 2 Wild Type Digest 3 Mutant Digest 3 Wild Type Observed Bands None None 3000-3300 and 16001500 None 6000-5500 4900 6000-2000 6000-2000 Table 6: Gel Electrophosesis Results Expected Bands None None 1612 and 3312 None 5851 and 1286 None 2874 and 2807 None Digest #1 Band Change No change 1612 3312 3312 Digest #2 Band Change No change 5851 1286 5851 Digest #3 Band Change No change 2874 2807 2807 Mutagenesis in pGLO Bacteria 10 Figure 1: Electrophoresis Gel Results Mutagenesis in pGLO Bacteria 11 Figure 2: pGLO plasmid Map Discussion This experiment yielded mixed results. Two of the experimental plates did not produce the expected results. We expected to see a white colony form on the plate containing wild type pGLO and ampicillin. No colonies formed on this plate, the most logical explination for this was that there was contamination when the condensation was wiped off of the plate lids prior to Mutagenesis in pGLO Bacteria 12 plating. This contamination could have introduced kanamycin to this plate and the wild type pGLO cannot grow colonies in the presence of kanamycin. Additionally, the plate containing wild type pGLO with ampicillin and arabinose did not produce colonies where we expected green colonies to form. Again, the most likely explination is cross contamination from the kanamycin containing plates when preparing the plates. This was definitely a case of human error as we didn’t think to use new wipes for each lid and had prepped our plates prior to the announcement that we should use separate wipes. In retrospect we should have started over with this step of the experiment as this oversight very likely skewed our results. The gel electrophoresis results were not the clearist. The first restriction enzyme appears to show two possible insertion sites. The brightest spot shows around position 3000 and a fainter spot around position 1500. This is in line with our expected insertion sites of 1612 and 3312. Because the spots are not as thick and as bright as restrict two it is less likely that the transposons inserted in these sites or that there was not sufficient DNA to produce a stronger result. The second restriction digest enzyme produced the clearist result with a very bright spot occuring around position 6000 and a faint spot occuring around position 2000. These results are in line with the expected transposon insertion points of 5851 and 1286. This would indicate that transposon inserted somewhere between the non-coding regions between AmpR and GFP and AmpR. The very faint bar around position 2000 is what we would expect for a transposon inserting in the GFP region, as our plate did not produce very many colonies the fainter lines is as expected because there was less DNA. The third restriction enzyme was not conclusive. It is difficult to determine if that transposon spanned a large area or if insufficient stain was used to yield the true results. It is Mutagenesis in pGLO Bacteria 13 possible that the transposon inserted around position 2800 but as there are no truly bright spots it is doubtful. As this is the non-coding region between AmpR and GFP we would not expect to see a strong reaction. It is difficult to say to what degree the results of this experiment were skewed by cross contamination in the initial set up of this experiment. It is also possible that the needed volumes of the components that went into the eight digest tubes was not calculated accurately leading to insufficient DNA used and an excess of water. Further we may not have used enough staining dye or allowed the reaction to procedure for long enough. When the gel was taken out of the reaction box it appeared as if the ladder was half way down the gel but it was a best guestimate. Mutagenesis in pGLO Bacteria 14 References Choi, K. H., & Kim, K. J. (2009). Applications of transposon-based gene delivery system in bacteria. Journal of microbiology and biotechnology, 19(3), 217-228. Leatherman, J. 2018. Genetics laboratory manual. Ann Arbor: XanEdu Pierce, B.A. 2014.Genetics a conceptual approach. New York: W.H. Freeman and Company
