Introduction double stranded molecule of DNA in
Introduction A plasmid is asmall circular double stranded molecule of DNA in a cell which is separatedfrom a chromosomal DNA; it can replicate independently (MEDLINE, 2012).Plasmids naturally exist in bacterial cells but are sometimes present in eukaryoticorganisms. Bacterial plasmids are commonly used as cloning vectors in theresearch of recombinant DNA; cloning is first performed using Escherichia coli(MEDLINE, 2009). After the construction of new plasmids, plasmid DNA isisolated from the E. coli cultures by using a method known as alkaline lysismethod.
Bacteria containing the plasmid is first cultured. Then, a sample iscentrifuged in order to concentrate cellular material (which includes DNA) intoa pellet at the bottom of the tube containing sample. They are identifiedaccording to their restriction enzyme pattern by using the technique of agarosegel electrophoresis. Under good conditions, after electrophoresis, lysis can beperformed on a colony of cells to enable detection of plasmid DNA in crudeextracts which allow for the analysis of various clones and aids thepossibility of screening. In this report, we will describe a simple method forthe extraction of plasmid DNA yielding plasmid DNA in a manner which is enoughto be digested by restriction enzymes. Plasmid DNA can be detected in a colonyof cells scraped from a plate or in a liquid (non-amplified) culture.Methods The extractionand purification of plasmid DNA from bacteria The bacteria weregrown overnight with the appropriate requirements and conditions at 37oC.
The bacteria are grown in an antibiotic containing medium which selects for thecontinued presence of the plasmid. When the bacteria are spread onto theantibiotic gradient plate and incubated, the various concentrations of theantibiotic in the plates and growth pattern show many significant molecularbiology principles which are as follows; (1) In a bacteria population grown ina culture (with nutrients), mutations may occur randomly in some bacteria. (2)The bacteria with better adaptation can develop mutation for antibioticresistance will survive on the plate containing the antibiotic. (3) somebacteria exhibit better resistance than others (Biotech) (Biomed, 2015).Antibiotic resistance in bacteria mostly occurs in plasmids.
Bacteria wascentrifuged (centrifuge was correctly equilibrated) and the growth medium wasremoved (supernatant discarded) to reveal the bacterial pellet. The bacterialpellet was resuspended in a solution of 50mM glucose, 25mM Tris (pH8.0), 10mMEDTA (pH8.
0). We observed that this solution became yellowish. A highlyalkaline solution consisting of 0.2M NaOH, 1% (w/v) SDS was added and the tubewas placed in ice. This is the lysis process.
The SDS (sodium dodecyl sulphate)is added to make the cell membrane more soluble and NaOH is added to break downthe cell walls and to convert the DNA from double stranded DNA to singlestranded DNAs by disrupting the hydrogen bonding between the DNA bases (Medline,2012). Upon adding the NaOH and SDS, the solution becomes clear. After solutioniii, containing 3M potassium acetate pH4.8, was added, tube was placed back inice during which the solution becomes cloudy. The addition of potassium acetate(solution iii) is to reduce the alkalinity in the mixture which does thereverse of what the NaOH and SDS does i.e.
to re-establish the hydrogen bondingof the single stranded DNA so that the single stranded DNA can be returned to adouble stranded DNA (Primrose, Twyman 2006). The solution was centrifuged againafter which a white precipitate was formed in clear solution. The supernatantwas then transferred, using a pipette, to a new microfuge tube. Two volumes of95% ethanol was added to the new microfuge tube containing supernatant and thencentrifuged. Then, supernatant was discarded. 1ml of ice cold 70% ethanol wasadded to the new tube, then directly discarded by pouring off. This step allowsthe salts to become more soluble while reducing the DNA solubility i.e toremove the leftover salts from the precipitation step (Primrose, Twyman 2006).
Then, the pellet is dissolved in 10mM Tris (pH7.6), 1mM EDTA (pH8.0) solution.20µl of solution from tube B was transferred to a newmicrofuge tube and a stock solution of RNA’se is added to the tube and thenstored at room temperature. The use ofrestriction enzyme (nucleases) For this part ofthe practical, we sued restriction enzymes (nucleases) to digest DNA isolatedfrom E. coli. We digested 5µl from the sample tubesB and C with 10 units of enzyme (ten fold excess of enzyme) in two new tubeslabelled tube BR and tube CR.
The two new tubes each contained DNA (B, C), 10xEcorR1 buffer, EcoR1 enzyme and water in equal amounts. Each constituent wasaccurately and carefully added using a pipette (and maintaining standard pipetteprocedure). The tubes containing enzymes were kept on ice when not in use sinceenzymes decay rapidly at room temperature. Then, tubes were left to incubate at37oC. Thetransformation of bacterial cells In this part ofthe practical, we applied the technique of bacterial transformation i.
e processof introducing DNA into bacterial cells. We used plasmids that can replicatewhen introduced into E. coli. But replication can only occur in circular formsof plasmids; linear forms of plasmids will not be able to replicate (Qin, Shen,Cohen 2003). Most of the plasmid used for this practical have genes forresisting antibiotic. Which means cells that contain a plasmid will haveantibiotic resistance and the rest will be sensitive. For this part of the practical,cells were inoculated with an overnight culture of CU-1 into L-Broth and shakenat 37oC(the cells were at the accurate growth phase for successful transformation.Cells were then poured into a falcon tube, placed in ice then centrifuged at 4oC.
The liquid was discarded to reveal pellet. Pellet was resuspended in 50mM CaCl2and left on ice. We also prepared DNA samples for the transformation process. Amixture of sample B with distilled water was added to a fresh Eppendorf tubelabelled diluted B. Three separate Eppendorf tubes were prepared containing;tube 1 0.1M Tris pH7.6, tube 2 diluted B and 0.
1M Tris pH7.6 and tube 3 EcoR1(digested B from BR) and 0.1M Tris pH7.
6. All tubes were then closed and placedin ice. Competent cells were then added to each of the three tubes and left onice. Eppendorf tubes were briefly transferred to a water bath at 42oCand placed back on ice. 1ml L-Broth was added to the tubes before transferplacing transformation reaction at 37oC. L-Broth is primarily usedfor the growth of bacteria (Geoff, 1994). The L-Broth medium, which is highlynutritional, is designed to enable the growth of the E. coli and transformationof the cells.
Since the cells are resistant to antibiotic, an enzyme which iscoded on the new plasmid (L-Broth) is added to the E. coli to activate theantibiotic resistance in order for gene expression to take place. We attemptedto calculate the colony forming units (CFU) of the plasmid cells in the plates.This is further explained in proper detail in the results section. Wedocumented the number of colonies formed in each plate and the CFU (Colonyforming units). Analysis ofextracted plasmid DNA using agarose gel electrophoresis For this part of the practical, we analysedthe isolated plasmid DNA from the previous experiments which were assayed usingagarose gel electrophoresis. The DNA travels towards the positive electrodes.
This is because when the DNA samples are loaded on to the gel, an electriccurrent is applied to the gel. Due to the phosphate groups in the backbone ofthe DNA, the DNA is negatively charged (Brown, 2010). Since the DNA has anegative charge, it makes more sense that the DNA will move towards thepositive electrode. Smaller DNA fragments will tend to travel faster than thelarger DNA fragments. The bands are formed due to the travel rate of thefragments which gather at a certain point. When the gel has been active longenough to separate the different fragment size, The DNA is stained using aSYBR-SAFE blue dye. We prepared an agarose gel with molten agarose poured intoa former.
After the gel is set, we moved the gel tray to the electrophoresistank and covered the gel in 1x TBE buffer. The six tubes A, B, C, B withRNA’se, B EcoR1 and C EcoR1 were prepared with TBE buffer (90mM Tris-borate,2mM EDTA), DNA and loading buffer (0.25% bromophenol blue, 15% ficoll 400)which were centrifuged to bring the contents to the bottom of the tube. Then,we transferred the prepared samples from the tubes to the agarose gel andapplied electric current to the gel (we started running the gel). We thenturned off gel and observed it using the trans illuminator. Results and Discussion E. coli in L-Broth and LB-amp plates observation (to detect how manycolonies were formed in each plate) L-Broth is primarily used for the growth of bacteria (Geoff, 1994). TheL-Broth medium, which is highly nutritional, is designed to enable the growthof the E.
coli and transformation of the cells. Since the cells are resistantto antibiotic, an enzyme which is coded on the new plasmid (L-Broth) is addedto the E. coli to activate the antibiotic resistance in order for geneexpression to take place. After overnight incubation, the LB-amp plates werecollected and observed. In our observations, we recorded that plate 1 had zerocolonies formed, plate 2 had about 300 colonies formed and plate 3 had about 9colonies formed. This simply means that the plasmid cells in the first platecompletely had no antibiotic resistance i.e. The plasmid cells in the second platehad the most antibiotic resistance i.
e. they possess the gene for resistingantibiotics and the plasmid cells in the third plate were almost completelysensitive to the antibiotic resistance (remember significant molecular Biologyprinciples which we stated earlier). Each distinct circular colony shouldrepresent an individual bacterial cell or group that has divided repeatedly, Weattempted to calculate the colony forming units (CFU) of the plasmid cells inthe plates but we were unsuccessful in finding it because we don’t have all thenecessary parameters required to calculate the CFU accurately.
Agarose gel results interpretation and explanation To denature the DNA chromosome, we exposed a crude extract to alkalinepH. After gel electrophoresis, we observed the gel using a trans illuminator.We also noticed that the different DNA fragments varied in size and speed(mobility).Lane C and CEcoR1 are a common result for good preparation of plasmids. There is not muchinterference in these bands. The fragments in the first well A are smallmolecules since they are able to travel far down to the anode (1.0kbp and0.
1kbp). They are a super helical form with high mobility. The DNA fragments inthe second well B are quite similar to well A.
The bands (4.5kbp and 2.0kbp)seem to be larger molecules as they moved only few steps away from the top(moderate mobility). The bands in well C are even larger fragments than theformer as they are about 10.0 kbp and 7.0 kbp in size. Bands in well B+also show moderate mobility at 4.
5 kbp and 2.0 kbp in size. Band in B EcoR1 (at3.0kbp) had average mobility. Bands in C EcoR1 (at 5.0 kbp and 4.
5 kbp) arealso quite large fragments of the DNA (low mobility). This means that most ofthe DNA fragments in this gel are very large molecules which resulted in theirlow mobility (at same amount of time). They all migrate towards the anodebecause most of the DNA is negatively charged and will therefore, be attractedtowards the positive charge. Band A contains RNA and more rapidly movingsupercoiled DNA because they smear the band that indicated circular DNA. It istoo big.
Same thing for bands B and C because they both also smear the bandindicating DNA and contain rapidly moving supercoiled RNA. Conclusion Although there isa list of methods available for the manipulation of DNA, we have, in thispractical, described a rather simple and reliable method for plasmid DNAextraction. This method has been successfully used in this experiment withoutany major challenge. Rapid DNA screening is almost always vital to choosecolonies which hold the recombinant plasmid DNA (and in a few cases, with theaccurate orientation of insert; plasmids DNA containing inserts of particularsize) (Davies, Jacob 1968).