Mordern molecular biology has discovered practical applications to understanding genetics.Depending on the nature of assays or laboratory techniques, scientists can carry out certain practicalgenetic applications like genetic engineering, diagnosis of genetic diseases and DNA sequencing ofspecies.
In this practical, alkaline lysis were used to extract plasmid DNA from 3 strains of E.coli (A, Band C). The plasmids were also added to restriction enzyme and RNAse followed by bacterialtransformation and gel electrophoresis to study their corresponding action on plasmid DNA.MethodAlkaline LysisIn the first practical, plasmid DNA from three cultures of E.
coli A, B and C were extracted by alkalinelysis. The basic principle of alkaline lysis is setting up reaction environment of pH ranged 12-12.5,which permanently denature chromosomal DNA but not plasmid DNA.Firstly, the cell cultures were centrifuged to separate the cells from growth mediums. The palletscontaining purely E. coli cells were suspended in solution I which contain 50mM glucose, 25mM Trisbuffer (pH8) and 10mM EDTA (pH8). The glucose balanced the osmotic difference between the cellsand the solution so to prevent cells from bursting. Tris solution had the role acting as physiologicalbuffer to stabilise the pH of the mixture of solution in the following steps.
Whereas EDTA wereprepared to chelate the metal ions like Ca 2+ and Mg 2+ . The process was necessary to disable thefunction of DNAse and prevent degradation of DNA molecules as the degradation requires these ionsas cofactors. The formed complexes were soluble in ethanol, therefore the metal ions could beseparated from the insoluble plasmid DNA complexes during ethanol precipitation.Secondly, the mixture were added with the solution II containing 0.
2M NaOH and 1%(w/v) SDSsolution. The SDS solution is a detergent that can solubilise cell membranes whilst NaOH can breakdown cell walls. By opening up the cells, the detergent bint to proteins with their high affinity andformed a protein-SDS complex.
The breakdown of hydrogen bonds denatured protein with alteredshapes. Similarly, the OH group in alkaline can breakdown of hydrogen bonds in chromosomal DNAand plasmid DNA. However, the plasmid DNA were able to renature when the solution wasneutralised because of its low molecular mass. The mixture was set in ice bucket for 5 minutes tolower its temperature. It is believed that the iced mixture could achieve better precipitation bydecreasing solubility of the protein-SDS complex.
Thirdly, the mixture were added with the solution III containing potassium acetate at pH 4.8. Thepotassium acetate neutralised the solution to allow plasmid DNA renaturation. Potassium ion alsoco-precipitated with the excess SDS and protein-SDS complex.
KDS (potassium dodecyl sulphate)were formed with an insoluble network. The denatured chromosomal DNA existed in the form ofsingle strand which were insoluble in high salt solution, the fragments were trapped by theprecipitate network. Whilst, plasmid DNA in supercoiled structure had small molecular size andsolubility in the solution which can pass through the network. The mixture was centrifuged and theprecipitates and insoluble molecules sink to the bottom of the tube.
A pallet was formed whichtrapped with cellular protein, high molecular weight DNA and high molecular weight RNA. Thesupernatant was transfer to another tube for ethanol precipitation and the pallet was discarded.After that, ethanol precipitation was performed for the supernatant. In the supernatant, due to thepresence water molecules in the aqueous buffer has high dielectric number, the water molecules inthe supernatant had higher resistant to the ionic bond forming in precipitation.
The water moleculesPak Lo U15755443form hydration shells around charged molecules and prevented the precipitate formation ofpotassium ions and phosphate ions on DNA backbones. The supernatant was therefore added with 2to 2.5 volumes of 95% ethanol to drive the precipitation.
The final concentration of ethanol in themixture would be around 70%. Ethanol has a low dielectric number, the supernatant at this pointhas lower overall dielectric number. It means the solution were less resistant to the ionic bondforming of precipitates. The phosphate groups on DNA molecules interacted with potassium ionsand precipitated in the solution. The solution was centrifuged so that to form a pallet with theprecipitated plasmid DNA. The pallet was added with ice cold 70% ethanol again to further removeany leftover supernatant, which the process could make the DNA pallet cleaner.
The pallets derived from E.coli cultures A, B, and C were dissolved in a TE buffer which is a buffermixed with Tris buffer and EDTA. The mixtures were labled as Tube A, B and C. Part of the thesolution part B was added with RNAse to make up tube B+.Transformation of Bacterial cellsPart of the extracted plasmid DNA from tube B and C were used for making tube BR and CR.
Firstly,the plasmid DNA from tube B and C were added with EcoR1 buffer and EcoR1 enzyme. EcoR1 is arestriction nuclease cleaving DNA at the recognition site with specific sequence, the enzyme werekept in ice to prevent protein decay when it was not being used. EcoR1 cleaves double stranded DNAwith the recognition site with sequence GAATTC and its complementary sequence CTTAAG. Thecleavage happens between the nucleotides G and A in the recognition site and create sticky endswith 4 nucleotides. The enzyme under a non-standard reaction condition can still mediate DNAcleavage. However, known as star activity, the cleavage of enzyme cleaves at a site that is differentto the defined recognition sequence. Therefore, the manufacturer buffer was used as it does notonly exclusively provide optimal reaction rate, it is more importantly to prevent the star activity.
Themanufacturer buffer includes Tris buffer to stable the pH at 7.5, Mg 2+ as cofactor and highconcentration of salt that increase ionic strength without inhibiting enzyme activity. The 10x EcoR1buffer was added orderly with water, plasmid DNA and the EcoR1 enzyme to make up to a finalvolume of 10 folds of the buffer added. At this stage, the tube BR and CR were complete and wereincubated at 37°C, which enzyme could be active under the temperature.
Next, tube B and BR were diluted with 0.1M Tris buffer at pH7.6 to make up a 50 folds final volumeof the mixture, and labelled with “diluted BR” and “diluted B”. The diluted BR and B were then usedfor E.
coli bacterial transformation. A negative control with only Tris buffer was also prepared. Afterthat, an unknown strain of E.coli was concentrated by centrifugation and suspended with coldcalcium chloride.
As cell added with CaCl 2 became fragile, cells were mixed by gentle swirling but notvortexing. The process had performed twice followed by adding cells into the negative control, tube”diluted BR” and “diluted B”. The tubes were heat pulsed in 42ºC for 2 minutes. Rahimzadeh,Sadeghizadeh and Najafi (2016) carried out experiment to conclude that transformation efficiency ofcells treated with and without heat shock were the same. However, another study (Panja, Saha andJana et. al.
, 2006) supported that calcium ion cooperates with negatively charged DNA andlipopolysaccharides on cell surface to form complex and the heat pulse step greatly lowered cellmembrane potential to facilitate plasmid penetration into the cells. In addition, the first experimentperformed the heat shock duration with only 45 seconds and the heating time might not besufficient for efficient plasmid penetration. Finally, the tube “diluted BR”, “diluted B” and negativecontrol were iced to recover to stable and non-fragile state. The tubes were then added with L-brothand incubated in 37 ºC to allow gene expression of plasmid. The cells in the tubes were thentransferred to three LB-amp plates which contained antibiotics. The plates would be observed laterwith characteristics acquired from the plasmid solutions treated with.
Pak Lo U15755444Agarose Gel Electrophoresis of Plasmid DNAAgarose electrophoresis can analyse DNA by loading DNA samples into a gel with electric field. Thenucleic acid with negative charged phosphate groups migrate to the anode of the gel which ispositively charged. DNA with larger size is hindered more as experiencing more resistance when thefragments get through the gel matrix. The agarose gel in the practical was prepared pouring themolten agarose into the gel former. A comb was inserted to allow the forming of wells in the gel forloading DNA samples. The molten agarose was set for approximately 20 minutes to solidify.
After thegel solidified, TBE buffer were poured into the former and covered the whole gel. The main purposeof adding the TBE buffer was to complete the electric current as mobile ion present. Duringelectrophoresis, water molecules were electrolysed to hydrogen ions at anode and hydroxide ions atcathode. The buffer also stabilises the uneven pH at anode and cathode as well as the gel.
After that,the DNA prepared from previous practical, tube A, B, C, B+, BR and CR were further diluted with TEbuffer and added with SYBR-SAFE. SYBR-SAFE is a nucleic acid dye which form complex with DNAthat emits visible light with 524nm under UV light. SYBR-SAFE has mutagenicity therefore werehandled carefully with safety protection on skin in the practical. Then the samples were loaded tothe gel wells for electrophoresis with electric field strength at about 100 volts and stopped when anyblue DNA band had travelled about 10cm.
ResultPlasmid DNA TransformationContent added with E.coli Colonies countPlate 1 Tris buffer 0Plate 2 Diluted B DNA 300Plate 3 Diluted BR DNA 5The LB-amp plate 1 added with only Tris buffer and competent E.coli cells had no colony formed. Theresult of the negative control indicates that the unknown strain of E.coli added into each platecontains no antibiotic resistance.
The 300 colonies formed in plate 2 were because plasmid DNA intube diluted B was circular which had entered and been expressed in the cells. In prokaryotes, linearDNA cannot be maintained and expressed, they were degraded by cell in general. It was notexpected that any colony would form in plate 3 as plasmid DNA were linearized by EcoR1. However,5 colonies were formed in plate 3. Possible reasons could be plasmid DNA B in tube BR were notcompletely linearized resulting to low proportion of circular plasmid remained in BR, linearizedplasmid DNA merged with the chromosomal DNA which was expressed with other chromosomalgenes or cells developed antibiotic resistance by natural mutation.Agarose Gel ElectrophoresisElectrophoresis analysis of the plasmid DNA samples can provide information to find out theapproximate size, quantity, purity and sequence in the DNA samples. Nucleic acids with differentmolecular size or weight form bands at different position. The picture of the resulting agarose gel isshown below with the labelled regionPak Lo U15755445.
RNAComparing tube B and B+, the tube B+ was added with RNAse to degrade RNA molecules. Therefore,tube B+ does not present a band in region 6. Whereas, tube B, as well as tube A,C, BR and CR,presented a band in region 6 which refers to the presence of RNA in the samples. However, tube BRand CR presented a paler bands than tube A,B and C. That indicates less quantity of RNA in the BRand CR as the samples were diluted from tube B and C.Chromosomal DNASmall amount of sample from tube C is present at the origin of the gel. That indicates molecules thatwere unable to pass through the gel network because of high molecular size or lack of molecularcharge.
Possible error could be careless pipetting in plasmid extraction that precipitates ofchromosomal DNA or protein were transferred with supernatant to tube C.Plasmid DNAPak Lo U15755446Comparing tube A, B and C, tube A does not present any band in region 1 to 5. That indicates therewas no plasmid DNA in the sample and E.coli strain A contains no plasmid DNA. Plasmid DNA in tubeB presents 2 bands at region 3 and 5 and plasmid DNA in tube C presents 2 bands at region 2 and 1.Plasmid DNA in prokaryotes are mostly in supercoiled circular form.
However, laboratory proceduresmay create nick DNA, which is the form of relaxed circular form of plasmid DNA. Figure 1 illustratesthat supercoiled form of plasmid has lower molecular size, though with same molecular weight asthe relaxed form, it experience less resistance and migrate faster to a lower position in gelelectrophoresis. Therefore, bands in region 2 and 5 of tube C and B are likely to represent theirsupercoiled forms of plasmids.
Whereas bands in region 1 and 3 are likely to represent their relaxedforms of plasmids.Figure 1 Retrieved fromhttp://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/index.html.EcoR1 cleavageComparing tube B and BR, BR was diluted and added with restriction enzyme EcoR1. Cleavagehappened to the circular plasmid DNA at 1 cleavage site. The cleaved circular DNA in BR opened upto a linear form.
The cleavage affected the shape but molecular mass of the linear plasmid did notchange. It experienced less resistance than the relaxed circular form, but more than the supercoiledcircular form. Therefore, the band representing the linear form of plasmid in BR in region 4, hadhigher position than supercoiled plasmid in tube B (region 5) and lower position than relaxedplasmid in tube B (region 3).Comparing tube C and CR, CR was diluted and added with restriction enzyme EcoR1. Cleavagehappened to the circular plasmid DNA at 2 cleavage sites. The cleaved circular DNA in CR wascleaved into 2 linear fragments. Unlike the cleaved plasmid in BR, the 2 fragmented linear plasmidDNA in CR travelled faster than both circular plasmid DNA in tube C as it has lower molecular mass.
Therefore, the fragments in CR formed 2 bands in region 3, which is a position lower than thecircular plasmids in tube C represented by the bands in region 1 and 2.Pak Lo U15755447Molecular weight and size of plasmid DNAThe molecular ladder of DNA from marker X is a molecular weight reference to dsDNA in linear form.Marker X contains linear DNA fragments with known molecular weight, it formed a ladder on the leftcolumn of the gel which can be referenced to the molecular weight of linear DNA in samples.Therefore, the molecular weight of plasmids from E.coli strain B and C were known by referencingthe bands of linearized plasmid DNA in BR and CR to the DNA ladder. Linear plasmid DNA of E.colistrain B was represented by tube BR with the band in region 4.
With reference to the DNA ladder,the plasmid DNA of E.coli strain B had molecular weight of 3 kilobase pairs.The the 2 fragmentedlinear plasmid DNA of E.
coli strain C was represented by tube CR with2 bands in region 3. Withreference to the DNA ladder, molecular weight of the 2 linear fragments of plasmid C were about 4.5and 5.
5 kilobase pairs. In other words, plasmid DNA of E.coli strain C had molecular weight of 10kilobase pairs.Discussion and ConclusionTo conclude, most of the macro molecules in samples were removed by alkaline lysis. And the RNAsesuccessfully degraded RNA in tube B+. However, small amount of macromolecules was remained intube C. As discussed, possible error could be careless pipetting of plasmid DNA supernatant inplasmid extraction, and should be avoided.
No single stranded linear form of plasmid DNA wereformed by irreversible denaturation of plasmid in alkaline lysis, which indicated the successfulcontrol of narrow pH range. Linearized plasmid B added to plate 3 grew 5 colonies of antibioticresistant E.colo.
As no circular plasmid DNA observed in electrophoresis of tube BR, the possibility ofnon-complete linearization of plasmid in tube BR is low. Further investigation is suggested on the 5colonies formed in L-amp plate. A gel electrophoresis can be performed to chromosomal DNA andthe plasmid DNA of the original strain and the transformed cells in the plate.
If the molecular weightof the chromosomal DNA in two cultures are different, and the difference is approximately same asthe molecular weight of plasmid B (3kilobase pairs), the linearized plasmid B was likely to havemerged into the chromosomal DNA during bacterial transformation.