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Construction of recombinant DNA molecules through DNA ligation and transformation is a very important basic tool in molecular biology. Transformation is used for cloning or to move DNA molecules around between strains. Bacteria are transformed for numerous different reasons. It includes expression of medically useful recombinant proteins such as insulin for treating a disease or vaccines for prevention of disease.
DNA Ligation and Transformation Introduction : Construction of recombinant DNA molecules through DNA ligation and transformation is a very important basic tool in molecular biology. Transformation is used for cloning or to move DNA molecules around between strains. Bacteria are transformed for numerous different reasons. It includes expression of medically useful recombinant proteins such as insulin for treating a disease or vaccines for prevention of disease. In this practical, the objective is to clone the DNA fragment encoding mouse beta-actin into the pGEM-T vector, followed by the transformation of ligated recombinant DNA molecules into the E.coli strain DH5α . The uptake plasmid DNA is further isolated from E.coli , selected against , verified by enzyme digestion , and finally visualized and analysed by gel electrophoresis. Materials : 2X Rapid Ligation Buffer for T4 DNA ligase , Linearized pGEM-T vector , PCR product , Control Insert DNA, T4 DNA ligase , Deionized water, SOC plate , Wizard miniprep Promega kit , Solution A , Solution B, Alkaline protease solution, Solution C, Solution D , 10X Digestion buffer , NcoI restriction enzyme, Sal I restriction enzyme , 1.2% agarose gel in TBE , Cyber Safe, 10X loading Dye, 1kb Ladder. Procedures: The PCR product ( mouse beta actin ) was ligated and incubated at room temperature with pGEM-T vector in standard ligation reaction , with an extra positive and an extra background ligation reaction as control . The E.coli DH5 α strain was made competent by ice treatment , and was added to the ligation mixture follow by a brief heat shock and incubation. The suspension of standard reaction , positive reaction and background reaction were cultured separately on SOC plate , incubated overnight at 37 C , and the number of blue and white colonies were tabulated. Each single blue colony and single white colony from Standard Reaction was transferred and cultured overnight at 37C in SOC plate. The recombinant plasmids were isolated from E.coli by a small scale preparation protocol using Wizard miniprep kit from Promega and stored. Each plasmid from blue and white colony were digested by NcoI or/and SalI restriction enzyme following by incubation at 37C for an hour. The vector pGEM-T and the insert was separated using Agarose gel electrophoresis and visualized under UV light box. Results: Table 1: Colony amount of different ligation reaction Colony Ligation Reaction White Blue Standard Reaction 2 0 Positive Control 0 1 Background Control 0 0 Table 2: Concentration and absorption ratio of purified recombinant plasmid DNA Plasmid form Concentration (μL/ng) Absorption Ratio 260/280 White Colonies 101.5 1.88 Blue Colonies 98.5 1.88 *Normal range of recombinant plasmid DNA concentration was yield. *Lane 1: Uncut plasmid from blue colony Lane 2: Nco-I digested plasmid from blue colony Lane 3: SalI digested plasmid from blue colony Lane 4 : NcoI and SalI digested plasmid from blue colony Lane 5 :Uncut plasmid from white colony Lane 6: Nco-I digested plasmid from white colony Lane 7: SalI digested plasmid from white colony Lane 8 : NcoI and SalI digested plasmid from white colony Lane 9 : DNA 1kbp ladder Figure : Digital image of plasmid restriction digests on 1kbp ladder Discussion: Blue white selection To select the transformed bacterial cell from the plate that have taken up the recombinant plasmid, the antibiotic (Amphicilin ) and a-complementation of β galactosidase is used. The antibiotic Amphicilin is a selection for antibiotic resistant colonies which have uptake the amphicilin resistant gene in vector plasmid. Thus, only cell with uptake of plasmid would be antibiotic resistant and survived as white colony. The β-galactosidase is a selection of bacteria colonies which have insertion of the DNA into plasmid which form recombinant plasmid. The recombinant plasmid with insertion of DNA would have disrupted Lac Z N-terminal , thus unable to have α-complementation with E.coli cell which expressing Lac Z C-terminal. The colony will remained white since no active b-galactosidase is produced to cleave the X-gal to produce blue product. Hence, white colony is observed for the cell that have the uptake of recombinant plasmid, blue colony is observed for the cell which have the uptake of non-recombinant plasmid, and no colony will be observed for cell which have no uptake of plasmid. From the result of table, the low amount/absence of white colony in standard reaction and positive control is due to the poor result of transformation. Unequal mixing of the ligation mixture with the E.coli cell during pipetting causing only small area of contact between the cell and the plasmid for transformation. This shows that uniform mixing of the reaction is important to facilitate the uptake of plasmid into E.coli cell. Restriction digestion analysis and gel electrophoresis From the result of figure 1 , in general, the bright band of 3kb is observed from the uncut plasmid and the plasmid with single restriction digestion . NcoI or SalI is only having one cut side in the plasmid thus producing only one 3kb band of vector plasmid. The band from lane 1-4( plasmid from blue colony) is slightly lower than the band from 5-8 ( plasmid from white colony ). This shows that the migration speed of plasmid from blue colony is slightly faster than plasmid of white colony , due to the difference in nucleotide base pair size . The plasmid of white colony which containing the recombinant DNA will have higher base pair size (>3kb) as compared to the plasmid of blue colony , thus will be having lower migration speed. Specifically, lane 1 and lane 5 show distinctive three bands which is due to the topoisomers of the uncut plasmid . The different migration speed of plasmid ( supercoiled >linear> relaxed ) causing three different bands to be observed. For lane 2, 3, 4, there are some extra bands ligther than 3kb band are observed due to the star activities of prolonged incubation time during the experiement . For lane 7, an additional 8kb band is observed due to imcomplete digestion of the plasmid from slightly higher concentration of enzyme added during experiment. For lane 4, although both NcoI and SalI restriction enzyme are added to the plasmid of blue colony , but there are still only one 3 kb band (vector plasmid) observed. This is due to the close proximity in the cut side between NcoI and SalI without insertion ,producing very small insignificant fragment size ( 45bp) compared to 3kb. As for lane 8 , there are 3kb band and 0.5kb band observed , in which the 0.5 kb fragment is the inserted DNA product. Conclusion: The inserted DNA product ( beta mouse actin ) is successfully isolated and verified throught the identification of 0.5 kb bands from the plasmid digestion image. The recombinant DNA is transformed into E. coli strain DH5 α. Hence, the transformed E.coli cell can be further cultured and lysed to produce many copies of purified DNA plasmid of beta mouse actin for other future application. References: Frey .M, Rall . S, Roth. A, Hemleben . V. ( 1980) . Evidence for uptake of plasmid DNA into intact plants ( Lemna perpusilla ) proved by an E.coli transformation assay . Z Naturforsch , 1980 . 35( 11-12) , 1104-6.
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