Blue-White Selection and Beta-Galactosidase Assay - Lab Report Example

Formal report: Blue/white selection and β-galactosidase assay 1. Introduction Blue/white selection is a technique used in detecting successfully ligated vector gene cloning. The foreign DNA can be ligated successfully into a vector, which is transformed into bacterial cells. The cells are grown in the presence of x-gal. If there is successful ligation, the colony will remain white, but if not, the colony would turn blue. This procedure is a simple and faster way of detecting the successful ligated genes. The organic X-gal compound contains galactose which is associated with a substituted indole, and is normally used to test the presence β-galactosidase. X-gal is an esters and indoxyl glycosides which produce insoluble blue mix parallel to indigo after being catalyzed by β-galactosidase enzyme (Brown, 2010). β-galactosidase, on the other hand, is a protein substance which is coded with lac Z gene of lac operon. Plasmids genes has antibiotic resistance genes which can be applied in the selection bacteria which contains recombinant plasmid which has clone DNA. The aim of this experiment was to obtain two types of transformed bacteria colonies, which are blue and white colonies using IPTG and X-gal. The blue colour indicates self-ligated plasmid that does not have DNA and the white colonies have bacteria which carries plasmid. ONPG was used to assay Β-Galactosidase to initiate the reaction. Cloning process is used to differentiate between the recreated vector and recombinant plasmids by plating with a selected marker. The colonies with transformed cells which have recombinant DNA molecules and those with self –ligated molecules after a blue white screening can also be differentiated. The antibiotic screening is the process of ligating DNA fragment into the resistant gene, the gene become inactive and the recombinant product can be identified through antibiotic resistance by transformants (Brown, 2010). 2. Aims The aims of this experiment were: 1) To reinforce the purpose of using IPTG and ONPG in genetic manipulation experiments that involve the Lac operon 2) To observe the differences in the extent of ONPG cleavage between blue and white colonies and to make inferences about why these differences exist 3) To observe the effects of IPTG on ONPG cleavage and explain these to reinforce understanding of how the lac operon works. 3. Methods Before the beginning of the experiment, two samples of E-coli culture with 25ml of sterile LB Growth medium with Ampicillin were labelled Lac+ and Lac- each. 5 blue transformant colonies were picked and the flask labeled Lac+ was inoculated, before swirling to suspend the bacteria. The culture was then incubated at 370C for 4 hrs. The same process was done on the white colonies on flask labeled Lac-. The optimum density at 600nm were also checked to make sure that it was between 0.5 and 0.7 by placing 1ml in a cuvette and putting in a spectrometer. Part A: Induction of β-Galactosidase At each point of time 3ml samples were removed from each flask and retained as the zero time point for the β-Galactosidase assay. Then they were placed on ice before reading the turbidity. 22 µl of IPTG were added into each of the 22ml culture so as to induce activity in β-Galactosidase. The culture were later incubated at 370C for half an hour, before removing 3ml from each culture and placing them in 13X100mm tube , immediately the OD600 were recorded. The remaining samples were incubated before removing 3ml from each of the culture and placing in 13X100mm. At different times turbidity were recorded from the spectrophotometer. Part B: Assay for β-Galactosidase in Blue and White Colonies After 1.5ml micro-centrifuge tubes in a rack were labeled, 2ml of the cultures form each sample were transferred into each assay group and the cell were spun for 5 minutes in a micro-centrifuge so as to pellet the cells. After discarding the supernatant, the pellets were resuspended in 500 µl phosphate buffer, before freezing to solids and thawing two times. Thereafter, 100µl of lysosome were added into each tube and incubated for ten minutes at 370C. 200µl of ONPG were added into each assay tube to initiate the reaction and incubated for 15 mins in 420C water. 4. Results Part A: Induction of β-Galactosidase Table 1: Absorbance Readings at 600 nm Absorbance Lac+/T-0 Lac- /T-0 Lac+/ T-1 Lac-/ T-1 Lac+/ T-2 Lac-/ T-2 OD600 0.233 0.185 0.225 0.230 0.265 0.240 Part B: Assay for β-Galactosidase in Blue and White Colonies Table 2: Absorbance Readings at 420 nm and 550 nm Absorbance Lac+/T-0 Lac-/T-0 Lac+/T-30 Lac-/T-30 Lac+/T-90 Lac-/T-90 OD420 0.045 0.303 0.249 0.086 0.309 0.074 OD550 0.047 0.263 0.058 0.027 0.018 0.016 5. Calculations a. The units of enzyme activity were based on the following equation: Miller Units = 1. Units of enzyme activity at 30 minutes (Lac+/T-30) Miller units = Miller units Units of enzyme activity (Lac-/T-30) = Miller units 2. Units of enzyme activity at 90 minutes (Lac+/T-90) Miller units Units of enzyme activity (Lac-/T-90) Miller units b. Table 3: Units of enzyme activity in the β-galactosidase-producing colonies/cultures (Lac+) versus (Lac-) Lac+ Lac- 30min 109.3 Miller units 28.1 Miller units 90min 174.5 Miller units 31.9 Miller units C. A graph of enzyme activity in the β-galactosidase-producing colonies/cultures (Lac+) versus (Lac-) Discussion 1. The graphs and data in a table At shown from the graph, as the β-galactosidase-producing colonies (lac+) increases those without (lac-) also increase. The Lac- E-coli mutants are not abundant as compared to Lac+. The graph indicates that the both grow on the agar media. They can be distinguished by the colour, but also Lac- is small in quantity. The data in the data shows that as the time increase, the Miller units for both Lac+ and Lac- increases which show that time is one of the factors in mutation (Casali & Preston, 2003). 2. Both blue white transformants colonies were inoculated using sterile inoculating loop so as to eliminate errors. It was then swirled to suspend the bacteria. Then the cultures were incubated for at an optimum temperature of 370C for 4 hrs to ensure maximum growth. The optimum density at 600nm were checked to make sure that it was between 0.5 and 0.7 by placing 1ml in a cuvette and before placing it in a spectrometer (Krebs, 2013). For the induction of β-Galactosidase, 3ml samples were removed from each flask and retained as the zero time point for the β-Galactosidase assay, before placing on ice for the assay. 22 µl of IPTG were added to each of the 22ml culture so as to induce activity in β-Galactosidase. Due to the sensitive nature of the mutation for the colonies they were incubated at an optimum temperature of 370C for half an hour, before removing 3ml from each culture and placing them in 13X100mm tube for recording (Gerstein, 2001). Assay for β-Galactosidase in Blue and White Colonies was prepared by transferring 2ml of the cultures from each sample into each assay group and the cell were spun for 5 minutes in a micro-centrifuge so as to pellet the cells. After discarding the supernatant, the pellets were resuspended in 500 µl phosphate buffer, before freezing to solids and thawing. Thereafter, 100µl of lysosome were added into each tube and incubated for ten minutes at an optimum temperature of 370C. 200µl of ONPG were also added into each assay tube to initiate the reaction and later incubated for 15 mins in 420C water as the water bath provides constant temperature. 3. Discussion The mixture contains ampicillin, LB, IPTG induce Lac z gene and x-gal. The plasmid and genes are placed in the host cells. The main reason is to use ampicillin as a marker and after screening the colour is observed. White and blue colour were expected because plasmid produce blue colonies in the presence of lac Z. Lac Z codes for β-galactosidase and in turn hydrolyses x-gal to blue colour. If the Lac Z region is interrupted by insertion of foreign DNA Lac Z gene and there would be formation of white colonies. The blue/white screening disrupts the process of α-complementation (Krebs, 2013). The vector has multicloning sites within lac z which can be cut by restricting enzyme such that the foreign DNA can be inserted in the lac Z gene, causing the interruption of the gene and the production of α-peptide. As a result, there would be no formation of β-galactosidase functional enzyme in the cell which had a vector with an insert. X-gal in the agar plate can detect the presence of β-galactosidase enzyme. The β-galactosidase cleaves x-gal to produce 5-bromo-4-chloro-indoxyl which oxidizes and dimerizes instantly to a blue insoluble colour. The blue colour colonies indicate the presence of vector with uninterrupted lac Z, thus no insert, but the white coloured colonies have unhydrolyzed x-gal which indicates that insert may be present (Palmer & Bonner, 2007). 4. A List of what have been learnt from these experiments - IPTG induces Lac Z gene to the vector to be expressed. Blue/ white screening cloning vetors uses lac operon. It carries the gene for β-galactosidase, with lac Z. The β-galactosidase enzyme catalyses the breaking down of lactose, which in turn acts as a source of food. It also degrades x-gal which is an artificial substrate which turns blue after being broken by β-galactosidase. -In summary, the colonies which are fed with x-gal and produces β-galactosidase will have a blue colour, but those which do not produce β-galactosidase will still be white even when fed with x-gal. The plasmids vectors used in blue/white screening have different cloning sites within the lac Z coding region. If the foreign DNA is successfully ligated into the multicloning sites lac Z is interrupted and the production of β-galactosidase is stopped. Thus for untransformed bacteria, there is no growth on the amp (Brown, 2010). - The bacteria which were transformed with the blue/white screening exhibited blue colour on amp with x-gal. The bacteria which were transformed with the blue/white screening with addition of DNA insert indicated white colour with x-gal. An additional IPTG agent was used as an inducer which assists in the expression of lac Z. It helps in the production of β-galactosidase which turns the colony blue even without lac Z gene expression. - The main use of lac operon is cloning. Lac operon has vital characteristics which make it to be controlled and the structural unit has β-galactosidase. When the host is grown on media sample containing IPTG inducer, and β-galactosidase substrate, X-gal, the colonies had blue colour (Brown, 2010). - When DNA fragment is inserted into Lac Z gene, the production of the enzyme is stopped and the enzymes remain white. Also of consideration is that some substances like lac Z gene has a mutation which can be complemented by another mutation carried by the host strain in another part of lac Z gene (Strachan and Read, 2010). Conclusion The plasmid and genes are placed in the host cells. Ampicillin was used as a marker and the colour was observed after screening. The white and blue colour were produced due to the that fact that the plasmid produced blue colonies in the presence of lac Z. Lac Z codes for β-galactosidase and in turn hydrolyses x-gal to blue colour. But if the Lac Z region is interrupted by insertion of foreign DNA Lac Z gene and there would be formation of white colonies. The errors in this experiment can be noticed from the control results which show divergence. The number of Lac- was very low, but there was a relative increase compared to Lac+. The errors can be due to self-ligation, incubation conditions, and human errors (Gerstein, 2001). The information learnt from this experiment included: 1. The purpose of using IPTG and ONPG in genetic manipulation experiments that involve the Lac operon 2. Examination of the differences in the extent of ONPG cleavage between blue and white colonies and to make inferences about why these differences exist 3. Studied the effects of IPTG on ONPG cleavage and reinforce understanding of how the lac operon works. This experiment has sharpened my skills that are essential when performing scientific research in future. I have also learned how blue/white screening technique works in detecting ligated vector gene cloning. I hope to use this knowledge in my future academic research. References Brown T. A., 2010. Gene cloning and DNA analysis: an introduction. Oxford, Wiley-Blackwell. Casali, N., & Preston, A., 2003. E. coli plasmid vectors: methods and applications. Totowa, N.J., Humana Press. Gerstein, A. S. (2001). Molecular Biology Problem Solver a Laboratory Guide. Hoboken, NJ, John Wiley & Sons. http://www.123library.org/book_details/?id=14782. Krebs, J. E., Lewin, B., Goldstein, E. S., Kilpatrick, S. T., & Lewin, B., 2013. Lewin's essential genes. Burlington, MA, Jones and Bartlett Learning. Metzler, D. E., & Metzler, C. M. 2001. Biochemistry: the chemical reactions of living cells. San Diego, Calif, Harcourt/Academic Press Palmer, T., & Bonner, P. L., 2007. Enzymes biochemistry, biotechnology and clinical chemistry. Oxford, Woodhead Publishing. Strachan T., Read A., 2010. Human Molecular Genetics, Garland Science Read More