Upon translating the gene sequence, presence of the RFP amino acid sequence confirmed the presence of the gene and successful PCR. Introduction The purpose of this lab was to use polymerase chain reaction (PCR) to amplify small amounts of DNA with two primers specific to two different genes: the red fluorescent protein (RFP) and green fluorescent protein (GFP). The DNA contains a specific sequence that is identified and annealed to by a specific primer.
The key idea in the experiment is to clone the Chromobacterium violaceium genes to be transfer to an Escherichia coli (E. coli) plasmid to see if the vio genes are the true genes that express the purple pigment. Cloning is the process in which a population is genetically produced identically to its mother cell by selecting only those sequences that correspond to protein-encoding genes [1] and to be inserted into a plasmid for gene expression. To use the genes of the bacterium for cloning, the genomic DNA (gDNA) must be extracted and used as a temple to examine the sequences as well as amplifying only specific genes that are desired. The use of polymerase chain reaction (PCR) is used to manipulate the DNA to an expression vector that would work to identify such sequences to produce the desired product.
Main reason on why PPP has been the only focusing pathway in this experiment is because the pathway that will produce xylitol in E. coli contain in PPP only. As stated by [10, 21], PPP is
Results Part 1: Effects of Heat on Bacterial Growth Table 1. Bacterial Growth Based on Heat 40 °C (Group 1) 55°C (Group 2) 80°C (Group 3) 100°C (Group 4) and (Group 5) Time (min) 10 20 30 40 10 20 30 20 30 40 10 20 30 40 Escherichia coli X X X
In the first part of the experiment, students will use observation to study the cause and effect of four different chemicals brought together and changed occurring. The four chemicals closely looked at are Calcium Chloride (CaCl2), Sodium Bicarbonate (NaHCO3), Phenol Red Solution, and Deionized Water (H2O). The chemical changes students will mainly look for is if there is a change of color, heat and/or light generated, gas produced and precipitate formation. Applying these principles in a practical manner are used to determine the underlying factors responsible for the particular observations. The second section of this experiment is conducting a series of controlled experiments to determine the cause of change in each observation made in the
From to the result we gain from these experiment, when we transfer microbes (E. coli) from slant to the deep agar by using stab wire, the shape of microbe form is filiform. Besides the shape filiform we get in the test tube, there is also some sort of crack on the agar that shows respiration occur in the agar. Usually, E. coli are remain viable but the oxygen availability is at the minimum rate that make the rate of growth of bacteria at slow pace. The result is taken after the test tube that contain the stab agar was incubated at 37° C for 24 hours. Microorganism shows great deal when it comes to oxygen requirement.
The purpose of this lab was to isolate the protein in cultured E. coli that emits a green glow. The green glowing protein observed is known as Green Fluorescent Protein, or GFP for short. To separate the proteins inside the E. coli, gel electrophoresis and hydrophobic-interaction chromatography was used. Hydrophobic-interaction chromatography is used to separate proteins that are hydrophobic, GFP is hydrophobic therefore did interact with the hydrophobic-interaction chromatography. Knowing that GFP is hydrophobic, it is inferred that it is in the column.
Thus, cross-contamination could be limited as much as possible by placing a cotton bud and some aluminum paper at the mouth of each conical flask. The pilot study involving Green Fluorescent Protein developed was to measure how much arabinose sugar is contained in five different drinks in the form of E414, gum Arabic, that is essentially made up of arabinose. The experiment did not work out as the arabinose content in the five drinks chosen was too little for the bacteria to glow. That failure generated the idea of varying the pH level rather than arabinose amount in order to indirectly measure, by comparison to the calibration, the pH of five other drinks. Those beverages should not contain E414 as it might affect fluorescence intensity, though insignificantly, but would still change the uncertainty of measurements.
Concluding the fact that the chloroplasts which are alike mitochondria have their own personal DNA, identical as prokaryotes, and organized in to operons, is a very good example or proof that they actually descend from the prokaryotic cells. Plasmids initially existed in the cells of the bacteria and also occurred in some eukaryotic cells. Very often, genes carried them in plasmids which provided bacteria with many genetic advantages, as was antibiotic resistance. Plasmids are not essential in order to let bacteria stay alive.
Bacterial Transformation: Effect of pGreen on the Amount of Bacterial Colonies on an Ampicillin Based Agar Plate Introduction: Bacterial transformation is the result that occurs when exogenous DNA is infused into a prokaryotic cell. The new genotypic makeup translates into a new phenotypic expression. In the case of the lab, the plasmid, pGreen was introduced to the experimental group.
A secondary sample that could have been tested, Sample 6, could contain an environment with -pGLO, Luria Bertani broth, and arabinose. This sample’s results should further illustrate the results of the lab, as due to a lack of +pGLO the bacteria should not grow, even with the arabinose sugar as a food source. Furthermore, due to a lacking of ampicillin resistance, again the bacteria should not show any growth as the cells will be considered “not transformed” and will not survive the plating. The addition of more samples would help broaden the understanding of genetic transformation and further illustrate the main point of the
Gram-negative bacteria appear red under Gram staining because they have thin cell walls that cannot hold the violet-iodine complex, but they can hold safranin. Reproduction: Bacteria reproduce asexually through a process called binary fission. This is when a cell (also known as the parent cell) divides into two identical cells (also called daughter cells) of identical DNA composition. The genetic material within the parent cell duplicates and moves to the opposite ends of the cell, invagination occurs, a cross wall forms and the cell splits forming two new daughter cells of identical DNA
Genetically modified Bacteria: Promise/Threat It is believed that over 3.5 billion years ago, bacteria-like organisms became the first inhabitants of the earth. Fossils from Greenland dating back to 3.86 billion years ago reveal what appears to be bacterial cells (Madigan 349). Bacteria are not only the oldest inhabitants of the earth, but also the most abundant and ubiquitous. They are found living in such unforgiving environments as Antartica and in geothermal vents deep in the ocean (Madigan 1, Willey 1).
The project has even served as a prototype for the Human Genome Project, illustrating the consequential impact such research could have on our world. As technology and knowledge of genetic engineering continues to advance, even larger and more ambitious projects can be undertaken; the research conducted on E. coli bacteria serves as the foundation and stepping stone for the small but substantial changes that can be made to any living organism - whether it be microbe, plant, or animal. The reengineering of such microorganisms has already been applied in testing for a cure to HIV, and this advance could soon lead to even more transcendent revelations. Consequently, the team is now seeking to recode the DNA segments of the synthesized E. coli into one unimpaired genome - testing whether the organism is capable of life and
Materials and methods Gene cloning and protein over-expression in E.coli: The gene encoding Mbgl was amplified from the genome of Methylococcuscapsulatus(bath)with the following primers: forward primer- TGGTTGGTTCATATGAGCAGATACGAGTTTCCAGAGCGATTCCTCand reverse primer-TATATATTAAAGCTTATAGTCCCGATCCAGGACGGCACCGTTGGTC (restriction site NdeI and HindIII are in italics and underlined). Prime STAR HS DNA Polymerase premix (DSS Takara, New Delhi, India) and the set of above-mentioned primers were used in the program consist of the following polymerase chain reaction (PCR) steps: (1) 95°C for 5 minutes (1 cycle), (2) 98°C for 20 seconds, (3) 50°C for 20 seconds, and (4) 72°C for 2 minutes (steps 2 to 4 was repeated for 35 cycles). The obtained PCR product was digested with NdeI and HindIII restriction enzymes and
