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Gene Expression of Promoter Lacy

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Gene expression of promoter lacY

[pic 1]

Constantine Hartofilis

BE 209 – Spring ‘17

Section B3 – Elliot

April 26th, 2017

ABSTRACT

Fundamental biology laboratory techniques were used in this experiment to identify an unknown promoter. The purpose of this lab was to create recombinant DNA vectors and study gene regulation by inserting them into E. coli bacteria cells. The methods used in order to develop these recombinant DNA vectors first required digesting the promoter sequence with restriction enzymes and then ligating the DNA fragments to form plasmids. These DNA products were then viewed using the gel electrophoresis technique. The plasmids were then amplified using polymerase chain reaction (PCR) technique with several primers to determine which promoter was present. Lastly, E. Coli colonies and different concentrations of inducer were inserted into wells and analyzed over time in a Time Analysis Experiment to observe Optical Density and Relative Fluorescence Units. The results of this step showed that the unknown promoter was lacY, which responds to the Lactose inducer. The Time Analysis experiment also showed the trend that expression of GFP was directly related to inducer concentrations. These findings depict the theory that inducers act to switch on promoter sequences placed upstream of genes. This regulation technique can be applied to create various amounts of a protein by inserting a promoter in front of a specific gene and inserting the developed DNA plasmid into cells.

INTRODUCTION

The human body is made up of trillions of cells, all used to perform various functions that allow for the overall organism to live and exist. All somatic cells in the human body contain the same genome or “genetic code” made up of twenty to twenty-five thousand cells. The genes serve to code for specific enzymes and proteins that perform various tasks and activities within the body. The purpose of gene expression regulation serves to allow cells to have specific functions and perform only specific tasks. Regulating gene expression is what allows for differentiation between the function of epithelial cells and osteoclasts. Sophisticated of gene expressions are seen though genomic research. Regulations pathways trigger developmental pathways, respond to external stimuli, and respond to dynamically changing environmental conditions and resource ability. Steps of gene expression can be broken down into transcriptional initiation, RNA processing, and post translational protein modification. One regulator steps often triggers the next and controls the rest of the pipeline in these complex regulatory networks (Ahmed, 2002). A large amount of effort is currently being devoted to characterize gene expression patterns and profiles to make standard information that can be widely known and applied by researchers all over the world.  

Various methods are available to quantitatively and qualitatively analyze gene expression regulation. Older methods include northern blots, nuclease protection and plaque hybridization that can only measure a single mRNA at a time and are difficult to automate (Ahmed, 2002). These techniques are platforms that provide some of the most accurate screenings of large sequences and evaluations of genes that have not previously been cloned or partially sequenced in a quantitative matter.

The objective of this experiment is to determine how three specific gene promoters are regulated. The promoter sequences will be inserted in front of the coding region of a green fluorescence protein, enabling the expressed gene to be easily viewed. This developed artificial DNA will be introduced into E coli cells and these cells will be exposed to various environments to determine how they survive and exist in them. By monitoring the fluorescence of the cells, conclusions will be drawn regarding the regulation of each promoter. Molecular cloning will be used to produce samples to use for experimentation. This protocol is essential for this experiment as well as genomic research as a whole. It allows for multiple copies of the same sequence to be present enabling for multiple types of experimentation on the same test sequence. The process of molecular cloning will be accomplished by isolating a particular sequence of plasmid DNA, inserting the sequence into a replicating vector, and obtaining multiple copies of the sequence for experimental use.

METHODS

In this laboratory experiment the fluorescent intensity of the cells were monitored by comparing the activity of each promoter developing a model on how these promoters are regulated. Promoter sequences were inserted in front of the coding region of a green fluorescence protein, artificial DNA was inserted into E coli cells and these cells were exposed to different environmental conditions. The methods used to complete this lab are common molecular cloning techniques.  

Isolation of Plasmid DNA

The first step of this process was to isolate the plasmid DNA from E coli cells using QIAprep Spin Miniprep kit. A liquid culture sample was obtained and 1.5 ml of the culture was placed into two separate 1.5 ml tubes and spun down at 8000 rpm for 60 seconds. Supernatant was removed from the tubes without disturbing the cellular pellet present at the bottom of the tube. The cellular pellets were then suspended in 250 μL of Buffer P1, 250 μL of Buffer P2, and 350 μL of Buffer N3. The tubes were mixed periodically between adding the buffers to avoid cell clumps. The tubes were then centrifuged for 10 minutes at max rpm to form a condensed pellet of chromosomal DNA, denatured proteins, cellular debris and SDS. After decanting the supernatant, the QIAprep spin column was centrifuged for 60 seconds and then washed by adding 500 μL of buffer PB, and then centrifuged for 60 seconds again. The spin column was then washed again by adding 750 μL of buffer PE and centrifuged for 2 minutes. The QIAprep spin column was then placed in a clean 1.5 mL tube along with 50 μL of Buffer EB and centrifuged for 1 minute. The tube with plasmid DNA was then placed in a -20 degree Celsius freezer for 1 week.

Restriction Enzyme Digestion/Analysis

Frozen tube of plasmid DNA was obtained and thawed using a bucket of ice. Three restriction enzymes digestions were developed: vector digest, single digest 1 and single digest 2. Vector digest contained 10 μL buffer, 10 μL plasmid DNA, 0.5 μL Xhol, 0.5 μL BamH1. Single digest 1 contained 10 μL buffer, 10 μL plasmid DNA, 0.5 μL Xhol. Single digest 2 contained 10 μL buffer, 10 μL plasmid DNA, 0.5 μL BamH1. Agarose gel was prepared using the gel solution and the form mold. Each lane of the gel contained a different sample: lane 1 contained 10 μL of the 2-Log DNA Ladder, lane 2 contained 10 μL of the Undigested Vector, Lane 3 contained 20 μL of Single Digest 1, Lane 4 contained 20 μL of Single Digest 2, and Lane 5 contained 20 μL of the Vector Digest. In addition, 5 μL of loading dye was added to each lane except lane 1. The samples were loaded into the gel and run for 45 minutes with a constant voltage of 120 Volts.

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