The lac operon is a transcriptional control of lactose metabolism in bacteria. The operon contains three transcriptional genes, lac Z, lac Y and lac A, which encodes for β-galactosidase, permease and transacetylase respectively. Lac P and lac O copes for the lac promoter and the lac operator, essential to the functioning of this operon. β-galactosidase converts lactose to allolatose, while permease allow lactose to be transported into the cell. Transacetylase does not have a role in lactose usage. In the absence of lactose, there is no allolactose, converted from lactose by β-galactosidase, to the active regulatory repressor, and thus the repressor binds to the operator and transcription is inhibited, as the RNA polymerase bound to the promoter is blocked. In the presence of lactose, allolactose binds to the repressor, rendering it inactive and unable to bind to the operator, allowing the transcription of the three structural genes. In this experiment, uvrA phr E. coli cells, repair-deficient mutant strain, were first exposed to one or two seconds or UV radiation. Since this strain lack both nucleotide excision repair and phtoreactivation to repair resulting pyrimidine dimers, mutations resulting from error-prone repair may occur in the lac operon. By plating out the mutated E. coli on MacConkey/lactose medium, we can determine whether the cells are lac operon mutants by assessing the colour of the colonies. MacConkey agar, acts as a pH indicator and stains bacteria that ferment lactose red (acidic pH). Bacteria that cannot use lactose will use the agar’s other constituent, peptone, instead, producing ammonia that gives a basic pH, and thus the colonies will appear pale. Thus, in our screening process, red streaked colonies are a... ... middle of paper ... ...lactose. By comparing the data from both the β-galactosidase activity assay experiment and the complementation experiment, we can determine the likely gene encoded in the lac operon, which had been mutated by UV exposure, and thus prevent lactose usage, in each of the two mutants. Thus, at the end of this project, we would have created and successfully isolated E.coli mutants that had a mutation in the lac operon, as a result of UV exposure, as well as identify the genotype of those two lac operon mutants. 1.1 AIM: 1. To isolate E. coli lac operon mutants via mutant screening using MacConkey/lactose and confirmation by growth in MacConkey/maltose plates 2. To determine the genotypes of two different E. coli lac operon mutants via β-galactosidase activity assay by spectrophotometry and the results of complementation test, with the introduction of various plasmids.
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
One bacterium was gram negative. It underwent four different tests. These tests were the EMB test (Eosin Mehylene Blue), the Sulfur Indole Motility (SIM) test, the Urease test, and the Simmon’s Citrate Utilization test. The EMB test checks for a bacteria’s ability to ferment lactose. This test is accomplished by placing the bacteria on Eosin Methylene Blue agar. The agar is selective for gram negative bacteria and those bacteria that can ferment lactose will have colored growth, usually a metallic green sheen.
Therefore colonies containing the non-recombinant pUC19 plasmid have a functional lacz’ gene appear blue on the agar and colonies containing recombinant pUC19 would have a non-functional lacz’ gene due to insertional inactivation and appear white on the growing medium.
In this experiment the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO and +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin. The plate with –pGLO, LB and ampicillin will show no growth and no glowing as no arabinose is present for glowing to be activated
The expression of lac operon in each tube equals the amount of beta-galactosidase produced. Therefore, by looking at the amount of beta-galactosidase under different conditions collectively is a good way to understand the function of inducers and repressors in supervising the expression of lac operon and the control of gene expression generally.
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
LI was first recognized in the 1960s when researchers found black children responding unfavorably to milk in their diets (Harrison 812). Research led to the discovery that lactose, the major sugar in milk and related dairy products, was undigestible in some people because they were missing the enzyme lactase. Lactase breaks down lactose into its component monosaccharide sugars, glucose and galactose. In people missing lactase, lactose passes undigested through the small intestine. In some people, the undigested lactose passes through the remainder of their systems with no ill effects. In others, however, the undigested lactose becomes viscous and ferments in the colon (Englert and Guillory 903). The thickness of the liquid and the fermentation cause painful cramping, gas and sometimes diarrhea. Besides not being able to digest lactose, these people suffer from malabsorption, which causes them to receive little or none of milk's nutrients (Houts 110).1
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
In this experiment, we perform a gel electrophoresis on the DNA. In this process, the enzymes were run through the gel electrophoresis to determine their relative sizes for each of them. The results of the certain DNA fragments are used in the final step, which is to construct a map of the DNA molecule. If we use different enzymes to cut DNA, then not every restriction site will be cut by all of the enzymes. The objective of this lab is to perform restriction enzyme of digesting plasmid DNA and constructing a map of plasmid from the results made from the experiment. Using this technique we understand what a DNA restriction enzyme is and how it works. In this process, the enzymes were run through the gel electrophoresis to determine their relative sizes for each of them. By following the experiment, we determined the positions of the restriction
By Molecular Approaches." Journal Of Dairy Science 96.8 (2013): 4928-4937. Academic Search Complete. Web. 12 Nov. 2013.
The synthetic A and B chains are then inserted into the bacteria’s gene for B-galactosidase, which is carried in the vectors plasmid. The vector for the production of insulin is a weakened strain of the common bacteria Escherichia coli, usually called E. coli. The recombinant plasmids are then reintroduced to the E. coli cells. As the B-galactosidase replicates in a cell undergoing mitosis the insulin gene is expressed. To yield substantial amounts of insulin millions of the bacteria possessing the recombinant plasmid are required.
Nuclease is one of the acknowledged proteins secreted by members of the genus Lactobacillus. After several evidences of extracellular DNAse activity, nucleases from several Lactobacillus species have been identified by two methods; sodium dodecyl sulfate polyacrylamide gel electrophoresis, coupled to in-gel protein renaturalization and nuclease assay [28]. It has been shown that nuclease activity over the DNA present in the luminal content, can lead to the formation of a set of diverse oligonucleotides, some of them with immunomodulatory properties [29, 30, 31].
Generally, lactic acid bacteria (LAB) can be defined as Gram positive, non-spore forming, catalase negative, devoid of cytochromes, acid tolerant, and facultative anaerobe group that produce lactic acid as the major end-product during fermentation of carbohydrates. According to carbohydrate metabolism, they can be divided into two main groups:
Hesiod’s Theogony and the Babylonian Enuma Elish are both myths that begin as creation myths, explaining how the universe and, later on, humans came to be. These types of myths exist in every culture and, while the account of creation in Hesiod’s Theogony and the Enuma Elish share many similarities, the two myths differ in many ways as well. Both myths begin creation from where the universe is a formless state, from which the primordial gods emerge. The idea of the earth and sky beginning as one and then being separated is also expressed in both myths.