M. Leprae Case Study

This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.

PCR is the amplification of DNA by denaturing, annealing, and extension of a DNA template. Specific sequences can be amplified using single-stranded DNA that complements the target sequences known as primers. This process heats DNA until the strands separate, then primers bind to the target regions. DNA polymerase enzymes and single base nucleotides (dNTPs) are used to synthesize new strands of DNA to the target sequence. The end product will contain large quantities of the target sequence (Bean et al. 2015). The most notable in phylogenic studies is the 16S rRNA gene, because of it’s highly conserved primer-binding site and hyper variable regions that provide species-specific sequences within bacteria and archaea (Kolbert and Persin 1999). This gene is a component of the 30S small subunit of prokaryotic ribosome’s and serves as the primary site of protein synthesis. (Woese and Fox 1977). The 16S rRNA sequence can be amplified and matched to national databases provided by the National Center for Biotechnology Information (NCBI) using software termed Basic Local Alignment Search Tool (BLAST) to find regions of similarity between biological sequences for bacterial identification. Thus, providing a cost effective and timely method when compared to biochemical

In the 1990s, further research in comparative genomics of bacteria and archea showed that in prokaryotic genomes, a majority of genes were acquired

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

There are many reasons for identifying an unknown bacterium. The reasons range from medical purposes, such as determining if the unknown could cause ailments in living things or knowing what microorganisms are needed to make antibiotics to other purposes such as knowing the exact microorganism has to be used to make certain foods. This experiment was done by applying methods in order to identify an unknown bacterium.

There are many reasons for identifying an unknown bacterium. The purpose of this exercise was to identify an unknown bacterium from a liquid culture. We chose our unknown bacteria from a rack of test tubes with several different species of bacteria inside. I wanted to pick an unknown bacteria with a number easy to remember so I pick the test tube labeled “745”. Procedures were followed as stated in the lab manual written by Dr. Pedro J.A. Gutierrez.

See Table 1 and Flow Chart 1 for results of Bacteria # 1 and Table 2 and Flow Chart 2 for results of Bacteria # 2.

“Ferdinand Julius Cohn (1828-1898): Pioneer of Bacteriology” Depatment of University of Memphis. 2004. Retrieved on January 16, 2014 from pnf.org: http://www.pnf.org/compendium/Ferdinand_Julius_Cohn.pdf

coli are picked and the plasmids are purified. The purified plasmids are used as a template for the sequencing reaction. The objective of the lab was to learn how to use the polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA gene from a bacteria colony, also be able to run an agarose gel to visualize the resulting PCR amplifications and extract the amplified DNA from the agarose gel.

Coli. The first standard E. Coli has no resistance plasmid while the second strain contains a resistance plasmid with genes protecting it from ampicillin. This standard E. Coli and pAMP (plasmid-Ampicillin) E. Coli were each streaked across plates containing the antibiotic and containing growth supportive Lurithea Broth. The purpose of this lab was to test their growth in each medium. Our hypothesis was that while the ampicillin resistant E. Coli would show growth in both LB and LB-AMP plate, the standard E. Coli would only grow in the LB plate for it contains no resistant plasmids against the

Our research on recombinant DNA mainly consisted of two experiments: Transformation and gel electrophoresis. In our first experiment, four microfuge tubes were given to us: pKAN DNA, pAMP DNA, unknown DNA, and a TE buffer without DNA. The two positive controls, pKAN and pAMP, consisted of an antibiotic resistance gene respectively to their name. The pKAN plasmid contained the gene resistance for kanamycin while pAMP carried the gene resistance for ampicillin. The negative control, TE, only contained buffer without DNA. The fourth tube was our unknown plasmid, which was either pKAN or pAMP; and by way of artificial transformation, we would be able to initiate the identification of our unknown plasmid.

Often scientists work with bacteria that do not come in a labeled test tube— for example, bacterial samples taken from infected human tissue or from the soil—and the scientist must then identify the unknown microorganism in order to understand what behavior to expect from the organism, for example, a certain type of infection or antibiotic resistance. However, because of the relatively few forms of bacteria compared to animals and because of the lack of bacterial fossil records due to their asexually reproductive nature, the taxonomy used to classify animals cannot be applied to bacteria (Brown 275). In order to classify unknown bacteria, a variety of physiological and metabolic tests are available to narrow a sample down from the fathomless number of possibilities into a more manageable range. Once these tests have been performed, the researcher can consult Bergey’s Manual of Determinative Bacteriology, a systematically arranged and continually updated collection of all known bacteria based on their structure, metabolism, and other attributes.

On June 25th, 2015 I chose the test tube labeled #19. This test tube contained an unknown bacterium, and the purpose was to determine the unknown bacterium by the end of the semester. Throughout the course, I ran a series of differential tests that would lead me to discovering the characteristics of my unknown. These tests that I will discuss in this paper are vital to understanding the biochemical mechanisms that different bacteria can perform, therefore helping me identify my bacterium based on molecular differences. During the course of this paper, I will refer to my unknown as unk#19. Also, I would note that aseptic technique was performed throughout the entire experiment and subcultures were regularly made.

And the unknown bacterial sample for Group 11 is… UNKNOWN. We were unable to identify our bacteria due to cross-contamination at some point during our research. This was first brought to our attention while running growth tests (Table 1). At that time, we discovered that most of the tests (EMB-lactose, PEA, Forms String in KOH) were showing that our sample was Gram-Positive, one test (Vancomycin) was showing that there was a Gram-Negative Bacteria growing.

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial