Beta-Galactosidase and Western Blot

The addition of 5-bromo-4-chloro-3-indoyl-β-thiogalactoside (X-gal) in the agar mix acts as an artifical substrate for the enzyme so it produces blue E.coli colonies when hydrolised by the β-galactosidase meaning those specific colonies do not contain the plasmid with the CIH-1 insert (non recombinant E.coli) (Coventry University 2016). Isopropyl-β-D-thiogalactoside (IPTG) an artifical transcription inducer of the lacZ gene is also added into the agar mix, binding to the repressor gene and inactivating it. Therefore when the CIH-1 gene is incorporated within the plasmid MCS and inserted into the E.coli

The GLAT enzyme itself belongs to the histidine triad super family and is a member of branch III. This enzyme shows specific nucleoside monophosphate activity and is a homodimer with each monomer containing a single domain comprised of 6 α- helices and a β-sheet which is formed by 13 antiparallel and 1 parallel strand. The mechanism of this enzyme is described as having ping-pong kinetics with the following two steps. In the first step the active site histidine attacks R-phosphorus of UDP-glucose which displaces glucose-1-phosphate and forms a covalent intermediate. The second step involves the previously formed intermediate reacting with galactose-1-phosphate to displace the histidine and produce UDP-galactose. (Facchiano, 103-104).

The enzyme that will be focused on is the Beta-lactamase enzyme. There exist many isozymes including Beta lactamase (Salmonella enterica subsp. enterica serovar Enteritidis str. 2010K-0284) and Beta lactamase (Thermoanaerobacter sp). The enzyme that will be reported on is the type TEM-1 Beta-lactamase (1).

To start the experiment two small plastic tubes were labeled negative pGLO and positive pGLO as the first step in this experiment. A pipet was then used to move 250 microliters of calcium chloride into each tube and the tubes were iced. Bacteria was added using a new sterilized loop each time to the positive and negative pGLO tubes. The loop was twisted around in the tube to ensure that the bacteria was evenly mixed into the solutions and the tubes were iced once again. Another loop was dipped into the tube containing the plasmid and it was removed when there was a visible slight film of residue. The plasmid was dipped into the tube that was labeled positive for the pGLO gene. The two tubes labeled positive pGLO and negative pGLO were iced

Plasmid map of pRSETB. Primers were designed to amplify via PCR to cDNA. The PCR product was digested with XhoI and EcoRI enzymes and ligated into the pRSETB plasmid. The pRSETB plasmid contains a T7 promoter region, is ampicillin resistant, inducible with Isopropyl β-D-1-thiogalactopyranoside IPTG, a molecular mimic of a lactose metabolite that triggers transcription of the lac operon, and has XhoI and EcoRI cut sites. The pRSETB plasmid is transformed into dH5α E. coli and plated on carbenicillin plates. Colonies are selected and grown on a carbenicillin plate while PCR is used to check that the plasmid that was up-taken was not

E. coli has an operon that codes for the breakdown of lactose. Within this lactose (Lac) operon, there are 6 total genes: LacI, LacP, LacO, LacZ, LacY, and LacA. LacI codes for the repressor that binds to the LacO operator (Hartl, 2011). The promoter region (LacP) is where RNA polymerase binds to begin transcription. LacZ codes for the enzyme (beta-galactosidase) that cleaves galactosides, glycosides containing galactose, into useable energy sources (Juers et al., 2012). For example, lactose is hydrolyzed into galactose and glucose. LacY codes for the membrane protein,

The purpose of this experiment was to find the range assay for determining the activity of the E. Coli enzyme, Beta-galactosidase. An enzyme assay is how enzyme activity is measured in a lab. It is useful to know the activity of a given preparation of an enzyme in order to know how much of the enzyme solution is needed for each reaction tube to produce a suitable reaction rate. Enzyme activity is also necessary in order to study enzyme kinetics and inhibition. To start, enzymes are biological catalysts that are made up of proteins and they help to speed up or catalyze a reaction.

The instructions tell the cell what to do and what traits to display. In this particular lab, pGLO plasmids was inserted into E.Coli bacterial colonies. This plasmid contained the bla gene, the araC gene and the GFP gene. These genes changed the bacteria by allowing it to be resistant to a particular antibiotic, Ampicillin and also to glow fluorescent green when in the presence of arabinose and when shown under a UV light. The bla gene codes for the beta lactamase enzyme which allows the bacteria to be resistant to Ampicillin, while the GFP gene codes for the protein that glows in the presence of UV light. Also, the araC gene codes for the protein that allows the GFP gene to produce the green glow protein only in the presence of arabinose. When the bacteria was in the presence of ampicillin and arabinose, the arabinose acted as an inducer which led the GFP gene to start producing GFP. This led to the bacteria glowing under UV light. This clearly shows gene expression as the GFP produced a protein after it was induced. I also learned from this lab that a cell’s DNA can be changed in order for it to produce different proteins. I also learned more about inducers, which bind to repressors to allow a gene to produce a protein. Furthermore, this lab also reinforced my knowledge of of how protein synthesis can be stopped or activated. Overall, this lab shows gene expression and is a primary example of how a cell’s traits can be

In analyzing the effect of 2-aminopurine on mutation frequencies of bacterial strain CC102 (ara∆(gpt-lac)5 thi rpsL/F’ lacZ-Y+A+proA+B+ , the bacterial strain CC102 was cultured in LB medium with 2-aminopurine and one LB medium without 2-aminopurine. The bacterial strain CC102 with and without the 2-aminopurine mutagens were diluted to 10-5 and 10-6 and were plated on LB plates with nutrients and sources of carbon compounds for growth. The undiluted bacterial strain CC102 was then plated on minimal medium with thiamine and lactose. In noting the genotype of the bacterial strain, there is a deletion in the lac gene as it is ∆(gpt-lac) and the lacZ gene is not functional, therefore β-galactosidase is not able to cleave lactose into the constituents of glucose and galactose. In table 1.1, when observing the number of mutant colonies on minimal lactose and single cell colonies formed on LB plates with no introduction of the 2-AP mutagen, the mutant colonies were not formed on the minimal lactose yet were formed on the LB nutrient rich plate. For example in looking at group 6 data, 163 colonies were formed on LB medium at 10-6 yet no colonies were formed on minimal lactose with no 2-AP. Since the lac gene and the lacZ gene need to be functional to cleave lactose, it would allow for growth on the minimal medium with lactose but since there was a deletion for the lac gene and lacZ gene was not functional, no growth occurred. However with the introduction of the

This was done by using DNA ligation and E.Coli transformation. We looked at agar plates to analyze which one of E.coli cell strains took up the vector alone of the vector containing the gene of interest. Four agar plates were used in this laboratory which were labeled Ligation, pGEM- T Positive Control, Competent Cells Negative Control, and Competent Cells Positive Control. Reagents were used such as DNA Ligase Buffer, SOC Media, Ampicillin, IPTG, and X-Gal. Ligation was performed using 2X ligation buffer, DNA Mix, T4 DNA Ligase and sterile dH2O. Then the transformation of E. coli occurred in which our 4 LB agar plates were prepared with the corresponding amount of IPTG, X-Gal and ampicillin gene. The next step was pipetting the content into the corresponding plates and SOC media was added.The transformation tubes consent were placed on the agar plates using a spreader. The plates were incubated at 37 Celsius and then will be stored at 4 degrees Celsius.The ligation plate was prepared by adding 100 µL of IPTG, 50 µL of X-Gal and Ampicillin. In the ligation plate, the expected color to be see was blue. However, in our ligation plate, we were seeing both blue and white colonies. Blue cells indicate that the cells take up the vector alone in the presence of IPTG and X-Gal due to Beta- Galactosidase expression. IPTG or

The results of this experiment suggest that an interaction is occurring between the EccA1 and the 4414 gene and that the regions responsible for the interaction are mainly N25 and N50. The N50 region of the 4414 gene might be where the interaction is occurring the strongest among the two regions. The CM –His –Trp –Ura plates with x-gal plate, Fig. 5, display the truncations on the left side as colored, indicating the production of b-galactosidase and that an interaction between the EccA1 and 4414 gene occurred. The columns in Fig. 5 that lack both EccA1 and 4414 gene and the top row that is missing only the 4414 gene show no color/b-gal production and therefore, had no interaction occur. The N50 and N75 cultures appear to be the most consistently dark out of all others; however, C25 does have a similar appearance to N25 and N50 indicating a similar production of b-gal and experience of interaction. Between N25 and N50, the consistent-dark color on the plates could potentially reflect an area among these regions responsible for the interaction. As for C25, a similar appearance to N25 and N50 could implicate the region as playing a role in the interaction.

which helps in the process of Lac operon. There are three genes which control the synthesis

The purpose of this experiment was to determine whether a point mutation on the lac- strain, CSH61, from the previous experiment is suppressed by amber suppressors, SuI, SuII, and SuIII. If the point mutation on the lac- strain was a nonsense mutation, a second mutation on the tRNA for that codon would be able to restore function of the lac gene by inserting an appropriate amino acid at the stop codon instead of terminating protein production. Therefore, the CSH61 cells could grow on lactose. If the point mutation was missense, no growth would be able to occur as the lac gene would not have its function restored.

Plasmids are small circular DNA molecules. They are not essential for survival of the host bacteria. Some carry genes that allow resistance to antibiotics (Anderson). Plasmid pUC18 is a circular DNA molecule. It contains portions of the E. coli Lac Z gene, which encodes for the first 146 amino acids of β - galactosidase. E. coli contains the Lac Z gene, which encodes β - galactosidase . The E. coli Lac operon digests lactose. Once E. coli is transformed with pUC18, complementation occurs. E. coli produces active β –galactoidase. The active β –galactoidase hydrolyzes the substrate, X gal , which is located on the agar plates.

Live Lactobacillus casei Shirota strain, cultured and tested in our laboratory, is added to the tank. The temperature of the tank is then reduced until the contents are at 37°C (body temperature). The solution is allowed to ferment in the tank for 6-9 days or until the numbers of Lactobacillus casei bacteria reach their ideal concentration.