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Micro-scale pAMP/KAN Recombinant Plasmid Production

 John C Gibson, Hunter Lyons

University Of Massachusetts, Lowell

Biological Sciences, Experimental Methods

Section 802A, Spring 2022

Micro-scale pAMP/KAN Recombinant Plasmid Production

Abstract

pAMP, short for plasmid DNA with a gene for ampicillin resistance, and pKAN, short for plasmid with a gene for kanamycin resistance, were cleaved by commercial grade mixture of BamHI(the first identified restriction enzyme from the H strain of Bacillus amyloliquefaciens) and HindIII(the third identified restriction enzyme from the d strain of Haemophilus influenzae) in this study. The cleaved fragments were ligated to produce recombinant plasmid DNA of pAMP/KAN. The dual antibiotic resistance efficacy with this novel plasmid for E. coli bacteria was tested successfully. The simple recombinant plasmid DNA was cloned and purified. Verification measures were performed in each step of digestion, ligation, and purification.

Introduction and Background

Recombinant DNA technology underwent significant growth since the 1970s with biological research and applications, such as the first isolation and cloning of mammalian β-globin “gene of interest” by Prof. Philip Leder at Harvard University[1]. The cloning techniques improved over time, such as the change from Ethidium Bromide with UV light in electrophoresis, used from the 1970s[7], to newer fluorescence agents and alternative light sources. The goal of this study was to establish a procedure utilizing current commercial supplies to produce small quantities of recombinant DNA and to test the product. This study aimed to give an update of materials and procedures appropriate for 2022. The cloned genes on plasmid products had very broad applications, including researchers establishing therapy of cardiovascular diseases[16] based on recombinant plasmids. The importance of understanding and grasping current material supply trends of this subject could not be overstated. 

Gene of interest, as mentioned, was essentially the gene studied with its protein originated from its transcribed mRNA from DNA and tested on life cells to determine the gene’s properties. Molecular cloning was the needed procedure to selectively bring the needed DNA, and subsequently its derived mRNA, to a proper quantity/dosage for experiments. Recombinant technology gave the bacterial clone growth selection advantage for the gene of interest, either by the recombinant plasmid carrier’s own selection advantage or by the payload gene’s. In this study, the KANr was tested for its antibiotic resistance property and therefore studied as a gene of interest that needed to be selected and cloned. pAMP was considered the carrier plasmid in this study because it provided the origin of replication for cloning. KAN lacked the origin of replication. It was hypothesized that simple recombinant pAMP/KAN was more easily produced than superplasmid or double transformation.

Overall, the cloning started with BamHI and HindIII enzyme digestion on stock plasmids because it was well known that this enzyme mixture preserved the KANr gene during cleavage of the carrier plasmid and the payload plasmid. In this study, SybrSafe was used as the fluorescence agent with blue light to verify cleavage progress and to verify the final product. The term, one-pot[9] ligation, could be perceived as inadequate precision by etymology, but the efficiency of the procedure made it suitable for this study to ligate the cleaved DNA fragments. In this study, a commercial grade, competent E. coli strain was used for transformation to incorporate the gene of interest into the living cells for cloning. And after the transformation, the inoculation loop was utilized for single-colony identical genome selection. Finally, the mini preparation procedure followed a textbook example to purify the plasmid DNA product samples.

Materials and Methods

Plasmid DNA Cleavage

BamHI and HindIII restriction enzyme mixture were utilized in this step, identical to textbook procedure[10], to cleave 2 plasmid DNA molecules, namely pAMP and pKAN. 5.5μL of pAMP stock plasmid and 5.5μL of pKAN stock plasmid were digested in 2 separate reaction tubes. The enzyme mixture was supplied by New England Biolabs(NEB for short), and stock plasmids were supplied by Carolina Biological Supply. The concentration of the stock plasmid DNAs were 0.2μg per 1μL. For each reaction tube, 2μL of BamHI/HindIII enzyme mixture was used. The reactants were suspended in a 1X restriction buffer, each tube 15μL by volume. Both digestions were then incubated at 37°C for 89 minutes. However, after the initial 30 minutes in incubation, a 5μL sample from each reaction tube was removed for electrophoresis analysis to verify digestion progress. The remaining 10μL sample of each reaction tube completed the full 89-minute incubation.

Gel Electrophoresis Of Digested Fragments

Following the textbook procedure[11], to verify digestion progress, a 5μL sample, drawn from each reaction tube at 30 minutes into the digestion, underwent electrophoresis in a 0.8% agarose gel and utilized SybrSafe as the fluorescence agent. The SybrSafe fluorescence agent was used to reduce toxicity of waste products. 1X TBE(Tris/Borate/EDTA) was used as the gel base as well as the electrophoresis solution[10]. 1μL loading dye was utilized as well as a standard λDNA HindIII digestion loaded for simultaneous electrophoresis. Electrophoresis was performed at 130 volt for 33 minutes. Transillumination was performed on the resulting gel with blue light.

One-Pot Ligation

The digestion enzymes in the digestion tubes were first heat-inactivated[12] by a 10-minute incubation of the digestion tubes at 79°C(slightly higher than textbook[12] inactivation temperature) after the 89-minute digestion. Then a 3μL sample from each of the completed digestions was drawn and combined in a single reaction tube to undergo recombination. The reactants were suspended in a 1X ligation buffer with 1μL of T4 ligase to form a 20μL total reaction volume. The ligation was incubated at 20°C for 18 hours.

Bacterial Transformation With Recombinant Plasmid

After completing ligation, 10μL of ligation volume was mixed into 100μL of commercial grade competent Douglas Hanahan strain(DH5α for short) of E. coli suspension to transform the bacteria. A heat-shock was administered at 42°C bath for 30 seconds followed by immediate cooling in an ice bath for 30 minutes, then the transformation suspension was mixed into 900μL of Super Optimal Broth With Catabolite Repression media(SOC media for short). The media with DH5α content was then placed in a shaking incubation chamber of 37°C at 250 RPM for recovery for 1 hour.  

Bacteria Plate Culture For Gene Selection

At the end of the recovery time, the recovery media with transformed DH5α were plated  onto 4 petri dishes, the first plate with only lysogeny broth in agar form(LB agar for short), the second plate with LB agar with added ampicillin, the third plate with LB agar with added kanamycin, and the fourth plate with LB agar with both ampicillin and kanamycin added, per textbook procedure[13]. Each plate received 230μL(larger volume than textbook procedure) of the recovery media with transformed DH5α content, and sterile spreaders were utilized to evenly spread the bacterial media across the entire agar surface. The 4 selection culture plates were incubated at 37°C for 18 hours.

Bacteria Selection And Gene Cloning

After 18 hours of growth, two isolated colonies in the plate containing LB agar with both ampicillin and kanamycin were picked up by sterile inoculation loops for identical genomes. Each identical-genome colony inoculated 5mL of cloning media in a separate culturing tube. Liquid LB with both ampicillin and kanamycin was used as the cloning media. The growth media was incubated at 37°C for 18 hours.

Plasmid DNA purification mini preparation

After 18 hours of cloning in the liquid media, the cloned DH5α E. coli cells were condensed into a pellet by centrifugation at 1400 RPM for 1 minute, per textbook procedure[14]. Each cloning media’s cell strain on its own sample tube in this step. Excess liquid drained. Then the cell pellet was loosened up with ice-cold 100μL GTE(glucose/Tris/EDTA mixture) solution and then lysed with 200μL SDS/NaOH(a mixture of SDS detergent and NaOH) to release plasmid DNA molecules from cell envelopes. The alkaline lysis was given 5 minutes to complete, then 150μL of ice-cold acidic potassium acetate KOAc was added to condense cell debris and to bring the pH level of the suspension back to neutral. A further wait time of 5 minutes was given, then condensed cell debris was precipitated out by centrifugation at 1400 RPM for 5 minutes and discarded. 

The suspended plasmid DNA from the supernatant was treated with added isopropanol alcohol, 400μL for each strain of sample, stood at 18°C for 2 minute, and then immediately underwent centrifugation at 1400 RPM at 0°C for 5 minutes. The 5-minutes centrifugation precipitated the DNA sample at the bottom of the reaction tube, which was further resuspended with added pure ethanol, 200μL for each sample. The DNA sample was further re-condensed with centrifugation at 1400 RPM for 3 minutes, and excess ethanol drained. The DNA sample was finally allowed to dry for 10 minutes, and then re-suspended in 15μL of TE(Tris/EDTA).

Plasmid DNA product verification - BamHI and HindIII cleaving

5μL of each of the 2 DNA samples were drawn and mixed into BamHI and HindIII restriction enzyme mixture in this step, per textbook procedure[15]. For each of the 2 digestion tubes and the control group of 5μL factory pAMP/pKAN mixture, 2μL of BamHI/HindIII enzyme mixture was added. All 3 digestion tubes and 2 non-digestion tubes of 5μL of each of the 2 DNA samples were suspended in a 1X restriction/RNase buffer, each tube filled with distilled water to reach 10μL by volume. All 5 tubes were then incubated at 37°C for 43 minutes. 

Plasmid DNA product verification - Electrophoresis

All 5 samples underwent electrophoresis in a 0.8% agarose gel and utilized SybrSafe as the fluorescence agent. 1X TBE was again used as the gel base as well as the electrophoresis solution. 1μL loading dye was utilized again, as well as loading the standard λ DNA HindIII on a separate lane for simultaneous electrophoresis. Electrophoresis was performed at 130 volt for 33 minutes. Transillumination was performed on the resulting gel with blue light.

Result

With the DNA digestion procedure on the commercially sourced pAMP and pKAN plasmids, the electrophoresis of the cleaved fragments in Figure 1(A) showed multiple bands of cleaved DNA in both experiment lanes of pAMP and pKAN of plasmids. The NEB lane contained all the 7 well-known λ(a bacteriophage) DNA fragments, among which, 4361 base pairs(bp for short), 2322 bp, 2027 bp, and 564 bp.

“pAMP” lane contained 2 bands and 1 smear. Using distance estimation, the first band in between the 4,361bp and 2,322bp markers was estimated to be 3,755bp, the second band close to the 564bp marker was estimated to be 784bp. There was a smear between the 4,361bp and 3,755bp. 

“pKAN” lane also contained 2 bands, the first band just slightly higher than the 2,322bp marker, estimated to be 2,332bp, and the second band just slightly lower than the 2,027bp marker, estimated to be 1,861bp.

Figure 1(B) showed multiple bands of cleaved DNA fragments in both experiment lanes of pAMP and pKAN of plasmids by the instructor. The instructor’s ideal gel’s NEB lane in Figure 1(B) also had 7 bands. Also in the instructor's ideal gel, both the pAMP lane and pKAN lane contained 2 clear bands. The 2 bands in pAMP lane were 3,755bp and 784bp. The 2 bands in pKAN lane were 2,332bp and 1,861bp. The instructor’s ideal gel’s pAMP and pKAN lanes did not have smears.

The remaining digestion, not used in the initial 30 minute digestion electrophoresis analysis, underwent ligation/recombination and transformed DH5α E. coli cells with recombinant plasmids in the bacterial transformation sub-procedure. The transformed E. coli colonies were shown in Figure 2(A)’s plating result, which displayed growth by media nutrients in all plates. Figure 2(B) summarized the relative growth per plate with their experimental control category.  

As shown in Figure 2(A) photograph, the LB Only plate, as the positive control group as indicated in Figure 2(B), had the most vigorous white lawn of growth of DH5α E. coli bacteria. The LB+AMP agar plate for ampicillin-only inhibition control had slight dotted white growth with approximately 40 colonies. The LB+KAN plate for kanamycin-only inhibition control also had slight dotted growth with approximately 40 colonies. The LB+AMP+KAN experiment group had the least growth, approximately 7 colonies of small white dots.

Two single colonies from the LB+KAN+KAN plate, after liquid culturing, cloning and undergoing DNA purification, produced the electrophoresis bands in the following Figure 3(a).

Figure 3 showed this experiment’s result of electrophoresis side-by-side with instructor ideal electrophoresis of simple-recombinant pAMP/KAN results. The SMP1- and SMP2- lanes in Figure 3(A) of this study experiment had a single band between 6557bp and 4361bp, estimated to be 5616bps. The SMP1- and SMP2- lanes in Figure 3(B) of the instructor ideal electrophoresis had a smear between 6557bp and 3755bp, estimated to be 5616bps by supercoil that had heavier molecular weight than the apparent travel distance. 

The SMP1+ and SMP2+ lanes in Figure 3(A) of this study experiment had 2 bands, one between 4361bp and 2322bp markers, estimated to be 3755bp, and another band slightly lower than the 2027bp marker, estimated to be 1861bp. The SMP1+ and SMP2+ lanes in Figure 3(B) of the instructor’s ideal electrophoresis also had 2 bands, one between 4361bp and 2322bp markers, estimated to be 3755bp, and another band slightly lower than the 2027bp marker, estimated to be 1861bp. Figure 3(A)’s pAK+ lane had 3 bands, estimated to be 3755bp, 2332bp, and 1861bp. Figure 3(B)’s pAK+ lane had 4 bands, estimated to be 3755bp, 2332bp, 1861bp, and 784bp.

Discussion And Analysis

The overall goal of this study could be restated as cleaving stock pAMP and pKAN as completely as possible to ligate them to create as much novel recombinants containing  both AMPr and KANr as possible. The 30-minute digestion analysis shown in Figure 1(A)’s pAMP lane had 2 bright bands of well known sizes, 3755bps[4] and 784bps[4], indicating intense digestion progress, which was desired for achieving the overall goal. pKAN lane also showed 2 bright bands of well known sizes, 2,332bp[4]s and 1,861bps[4] respectively, indicating intense digestion progress as well. The band locations at 3755bps, 784bps, 2332bps, and 1861bps made sense because typical Bam and Hin group restriction endonucleases cut DNA molecules on average once every 4000 base pairs[5][6]. The combined BamHI/HindIII therefore cut average 2000 bps fragments as expected. The fact that this experiment’s band patterns matched the instructor’s ideal gel band patterns of Figure 1(B), namely 3755bps and 784bps in pAMP lane and 2,332bps and 1,861bps in pKAN lane, reaffirmed the success of enzyme digestion. 

Furthermore, figure 1(A)’s pKAN lane indicated that digestion was complete within 30 minutes by the total absence of the original 4193bp plasmid molecules between the 23,130bps marker and the estimated 2332bps distance, which was identical to instructor ideal gel’s pKAN lane of Figure 1(B). Figure 1(A)’s pAMP lane’s smear near the 4361bps mark implicated incomplete digestion at the 30 minute time. The undigested 4539bp molecules traveled slightly further than the 4361bps mark by supercoiling, which reduced the molecule’s apparent size. However, the smear of the 4539bps molecules was relatively faint compared to the digested bands of 3755bp and 784bp. This contrast meant that, by the end of 89 minutes of the full digestion period, the pAMP digestion should have also been complete. 

The results of all 4 culture plates in Figure 2(A) all made sense because the pAMP and pKAN digestion was confirmed to be successful by electrophoresis, so the ligated fragments must have transformed DH5α to give rise to antibiotic resistance in LB+AMP and LB+KAN and LB+AMP+KAN plates. For the LB Only plate, the vigorous growth in this control group meant that LB medium was fit for the experiment, and that the severely inhibited growth in the LB+AMP+KAN experiment group was due to antibiotic inhibition, not due to poor nutrient supplies. The partially inhibited growth in the LB+AMP plate meant that the experiment’s ampicillin was effective, and hence the severely inhibited growth in the LB+AMP+KAN was not the result of kanamycin bactericidal action alone. The partially inhibited growth in the LB+KAN plate meant that the kanamycin was effective, and hence the severely inhibited growth in the LB+AMP+KAN was not the result of ampicillin’s inhibition action alone. The 3 control groups together showed that the LB+AMP+KAN plate exerted high selection pressure favoring cells with both AMPr and KANr genes, and that the isolated colonies in the LB+AMP+KAN plate must had both AMPr and KANr genes in each cell. It was not reasonable to attribute the dual antibiotic resistance to either a single transformation of AMPr or KANr in any individual cells because both ampicillin and kanamycin were shown to be effective and fit for experiment by the 2 mutually assured control groups of LB+AMP and LB+KAN. The growth in the LB+AMP+KAN plate thus unquestionably indicated the success of plasmid transformation of both  AMPr and KANr genes into individual cells, as expected.

Considering that the efficiency of plasmid transformation of bacteria decreases with the increase in plasmid size[8], the pAMP/KAN novel plasmid of 5616bps (3755bps and 1861bps combined) was more likely to enter the E. coli cell than the superplasmid pAMP/pKAN of 8732 bps (4593bps and 4193bps combined). Comparing the chance of a single pAMP/KAN novel recombinant DNA plasmid entering the cell to the chance of dual plasmids of both the regenerated pAMP and pKAN entering the cell, the single event of pAMP/KAN entering the cell was more likely. The resulting electrophoresis pictures in Figure 3(A) demonstrated such expected likely results by the total absence of superplasmid of 8732bp band in any lane and the total absence of 2332bp band in the SMP1+ and SMP2+ lanes. In Figure 3(A) the simple recombinant of 5616bps appeared as a clear band in SMP1- lane between the 6557bp and 4361bp markers, which made sense by the simple ordering of the molecular sizes. If double transformation had occurred, the cloned pKAN would have been digested as 2332bp and 1861bp bands. The absence of the 2332bp band in the SMP1+ and SMP2+ lanes rejected the interpretation of double transformation giving the dual-antibiotic resistance seen in Figure 2(A)’s LAB+AMP+KAN plate.

The same band of 5616bp in SMP1+ lane appeared fainter in the SMP2- lane, but the appearance did not alter the inferred molecular size by the band travel distance. The fact that the instructor’s ideal gel band pattern in Figure 3(B) being identical to this experiment’s Figure 3(A) meant that simple recombination ligation unmistakably occurred in this study experiment.

Conclusions

The goal of this study was to establish a procedure to cleave the factory stock pAMP and pKAN as completely as possible to produce as much recombinant plasmid DNA with AMPr and KANr genes together in plasmid in a living cell for cloning to a micro scale. And as shown in discussion, this study experiment successfully produced the simple recombinant pAMP/KAN plasmid , which was 5616bps in length, 3755bps(pAMP) and 1861bps(KAN) combined. Overall, the goal of this study was achieved. The micro scale production steps in the experiment followed the well known procedures as much as possible and the desirable results were achieved. The cleavage of the stock factory plasmids was considered complete, the one-pot ligation created the needed recombinant, and the inoculation loop selection of bacteria picked out the needed simple recombinant colonies. The improvements in the future could be the loading for electrophoresis. More practice would result in more complete DNA material loading in the loading well and brighter electrophoresis bands.   

References

[1] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 143.

[2] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 495.

[4] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 451.

[5] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 111.

[6] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 144.

[7] Aaij, C, and P Borst. "The gel electrophoresis of DNA." Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, vol. 269, no. 2, 31 Dec. 1971, pp. 192-200.

[8] Szostková, Monika, and Dana Horáková. "The effect of plasmid DNA sizes and other factors on electrotransformation of Escherichia coli JM109." Bioelectrochemistry and Bioenergetics, vol. 47, no. 2, Dec. 1998, pp. 319-23.

[9] Potapov, Vladimir, et al. "Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly." ACS Synthetic Biology, vol. 7, no. 11, 2018, pp. 2665-74.

[10] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 443-450.

[11] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 449.

[12] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 453-454.

[13] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 463-467.

[14] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 427-429.

[15] Micklos, David A., and Greg A. Freyer. DNA Science - A First Course. 2nd ed., Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2003, p. 489-491.

[16]Williams, Paul D. Williams D., and Paul A. Kingston A. Kingston. "Plasmid-mediated gene therapy for cardiovascular disease." Cardiovascular Research, vol. 91, no. 4, 1 Sept. 2011, pp. 565-76.