Chromobacterium Violacein Test Lab Report

Found in subtropical environments, Chromobacterium violaceum (C. violaceum) is a gram-negative bacillus that has a single polar flagellum with one or two lateral flagella [2]. The organism has an operon that is composed of violacein (vio) A, B, C, D, and E genes. The present of violacein, a purple pigment and a natural antibiotic, plays a huge role in protecting the bacteria from reactive species that act as an antioxidant toward the bacteria. Sometimes, the appearance of the purple pigment would not appear if one or more of the vio gene(s) are block. The appearance of a non-purple pigment will results if not all the vio genes are turned on, which leads to no production of violacein. The antibiotic is also known to be used as a treatment of

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. There are three major parts in isolating gDNA: 1) lysing cells, 2) separation of DNA, and 3) recovery of purified DNA. In other terms, the purification and amplification of a specific DNA sequence by DNA cloning in bacterium would allow the production of many copies of the same sequence which can then be inserted into a plasmid vector to be expressed the desired genes

First, a culture of the bacteria will be grown in a broth and the gDNA will be extracted from it. Next, the gDNA will be used as a DNA template to amplify only the vio CDE gene with through a series of PCR and nanodrop to check purity and contamination of the sample. Then, the genes will be made into an expression vector through Gateway cloning in which the vector can be then be transformed into colonies where the vio pathway can be manipulated to produce their desired pigments. Because the goal of this experiment was for a Chromobacterium violaceum bacteria to turn the violacein (a dark violet pigment) into light purple, a pathway involving five different enzymes vio A, B, C, D, E were needed. The process requires multiple cloning sites, the insertion of a gene of interest, using restriction enzyme digestion, and ligation in order to manipulate the expression of the desired gene. Finally, the genes were transfer into E. coli organisms that to express the purple pigment known as