Download MHP Lab 6 - Transformation and Transcription

Lab #6
Name: _________________________________________________
Part 1: Bacterial Transformation
Part 2: Transcriptional Regulation
Part I : Bacterial Transformation
INTRODUCTION
In this lab you will perform a procedure known as a genetic transformation. Remember that a
gene is a piece of DNA that provides the instructions for making (coding for) a protein that an
organism requires for appearance or function. Genetic transformation literally means change
caused by genes and it involves the insertion of a gene(s) into an organism in order to change the
organism's trait(s). Genetic transformation is used in many areas of biotechnology. In
agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically
transformed into plants. In bio-remediation, bacteria can be genetically transformed with genes
enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning
to be treated by gene therapy; that is, by genetically transforming a sick person's cells with
healthy copies of the gene involved in their disease.
You will use a procedure to transform bacteria with a gene that
codes for a Green Fluorescent Protein (GFP). The real-life
source of this gene is the bioluminescent jellyfish Aequorea
victoria. GFP causes the jellyfish to fluoresce and glow in the
dark. Following the transformation procedure, the bacteria
express their newly acquired jellyfish gene and produce the
fluorescent protein that causes them to glow a brilliant green
color under ultraviolet light.
In this activity, you will learn about the process of moving genes
from one organism to another with the aid of a plasmid. In
addition to one large chromosome, bacteria naturally contain one
or more small circular pieces of DNA called plasmids. Plasmid
DNA usually contains genes for one or more traits that may be
beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth, which
creates the opportunity for them to share these beneficial genes. (Note that the bacteria don’t
know that they are picking up beneficial genes.) This natural mechanism allows bacteria to adapt
to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the
transmission of plasmids.
You will be provided with the tools and a protocol for performing genetic transformation in
Escherichia coli. This transformation procedure involves three main steps. These steps are
intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the
cells to express their newly acquired genes. Many species of bacteria have special membrane
proteins for the uptake of DNA from the external environment. E. coli does not appear to have
these types of membrane proteins; however, placing E. coli in a relatively high concentration of
calcium ions and performing a procedure called “heat shock” will stimulate these cells to take up
pieces of foreign DNA.
Read the lab exercise and follow the directions carefully. Completion of the lab will take 2 lab
periods (or 1 lab and 1 class). In the second lab period you will analyze your results.
You will practice using sterile technique, as instructed at the beginning of lab session, before you
do the experiment. When culturing bacteria, you must not introduce other, contaminating bacteria
into your culture. Potentially contaminating bacteria are ubiquitous; they are found everywhere
(including on the bench top and on your hands). It is especially important to keep the inoculation
loops, the pipette tips, and the surfaces of the agar plates must not touch or be touched by any
contaminating surface.
PURPOSE
To transform bacteria with plasmid DNA and analyze growth on agar plates containing either
ampicillin alone or ampicillin and arabinose.
MATERIALS AND PROCEDURES
1. Follow the procedures in the “Transformation Kit-Quick Guide” provided in lab.
2. The plates will be incubated for 24-48 hours, and then placed in a refrigerator to slow the
growth of the bacteria. You will than observe the plates in the next lab period to collect your
data.
3. Complete your lab report: Answer the questions and fill in the table in the space provided
below. Complete the Hypothesis, Predictions, and Experimental Design sections during the
first lab period. The Results section will be completed after we analyze the data next week.
PRE-LAB QUESTIONS
What is ampicillin? What is arabinose? What are they used for when growing bacteria on LB-agar
plates? NOTE: LB is the nutrient mixture that is added to the plate agar to feed the bacteria.
Based on the above answers, prepare and complete the table below to indicate what you predict
will happen on each of the four agar plates. (Will E. coli grow on these plates? Will the E. coli
have any special properties compared to wild type?)
Plate
Plasmid?
LB/amp
LB/amp/ara
LB/amp
LB
+DNA
+DNA
-DNA
-DNA
Growth (G)
No Growth (NG)
Other properties?
1. What is/are the independent variable(s) in this experiment?
2. What is/are the dependent variable(s)?
3. Which plates will serve as control plates? Do you expect cells to grow on these plates? Why
or why not? What is the purpose of these controls?
4. Define plasmid.
Part 2: Transcriptional Regulation
INTRODUCTION
We know that DNA is the genetic material for life. DNA is transcribed to make RNA, and the RNA
serves as a template for protein synthesis. In the class, you are learning that this description of
the process is over-simplified. In fact, there is regulation of every step in the process, right down
to how long the protein remains stable inside the cell. In this section, we are going to examine
how transcription can be regulated – How does the cell decide if RNA will be made from a
particular gene template. Although we will not do the actual experiments to test transcriptional
regulation in a eukaryotic system, you will design a “mock” experiment and I will provide you with
data to analyze.
The scenario:
Imagine you are working in a lab. The student who was working there before you discovered a
new gene – NSCC1 (Figure 1). She cloned the whole coding sequence (dark blue box), as well
as the upstream promoter region (left of the blue box). The promoter region of a gene is what
determines whether or not a gene will be used to synthesize RNA. The process of synthesizing
RNA based on a gene template is called “gene expression”. The promoter region contains
specific sequences that bind proteins known as transcription factors. Binding of transcription
factors to the promoter signals the proteins that transcribe all genes to express that particular
gene (see Chapter 17 of your textbook).
After sequencing the DNA clone, the student found 3 E-box sequences in the promoter (light blue
boxes). The sequence of an E-box is CANNTG, where the Ns can be any nucleotide. The
interesting part about this sequence is that it may bind to transcription factors found only in
skeletal muscle cells, such as MyoD. Your task is to figure out whether or not this is the case for
this particular gene.
Figure 1 – The new gene.
possible E-boxes
transcriptional start site
TATA box
unknown gene – NSCC1
promoter
In order to determine this, you first ligated the promoter sequence of the new gene into the pGL2
reporter vector, shown in Figure 2 (don’t worry about the details of this – we will come back to it in
the DNA Technology section). So, now your promoter sequence is upstream of the luciferase
gene in the plasmid, instead of NSCC1. These plasmids can be transfected into mammalian cells
using specific transfection reagents. If the cells that you transfect have the transcription factors
that bind to the promoter region, and these factors can bind to the plasmid DNA, they will activate
transcription of the luciferase gene, thus making luciferase protein. Luciferase is an enzyme from
fireflies that catalyzes the breakdown of luciferin to generate light. After the cells are transfected,
the proteins in the cells are given time to find the plasmid and activate transcription of the
luciferase gene. Protein is isolated from the cells - the amount of protein isolated can be
measured using a specific protein quantitation reagent and a small sample of the isolate. The
activity of the luciferase enzyme is measured by adding luciferin to a sample of the protein extract
and measuring the light emitted using a luminometer. The amount of luciferase activity is directly
related to how well the promoter is able to activate expression of the luciferase gene – promoter
binds transcription factors, gene is expressed (RNA is made), RNA is made into protein.
You have three cell types available to you – skeletal muscle cells (C2C12 cells), kidney cells
(COS-1) and fibroblasts (3T3). The skeletal muscle cells have muscle-specific transcription
factors such as MyoD. MyoD can bind to the E-box sequence and activate transcription of certain
muscle-specific genes. COS-1 kidney cells and 3T3 fibroblasts do not have the MyoD
transcription factor and therefore do not activate transcription of these muscle-specific genes.
Keep in mind that you have a plasmid that will make MyoD protein in fibroblasts.
Figure 2 – pGL3 reporter plasmid.
luciferase polyA
promoter
ampicillin resistance
MATERIALS
C2C12 cells
COS-1 cells
3T3 cells
(assume you have all the things available to grow the cells)
Tranfection reagent
pGL3 plasmid
pGL3 plasmid containing the promoter
EMSV plasmid containing MyoD cDNA (can express in fibroblasts)
Protein extraction buffer
Luciferin
Luminometer
Protein quantitaton reagent
Spectrophotometer
(assume you have tubes available to you)
PROCEDURE
Let us begin with the hypothesis that the gene will only be expressed in muscle cells. How are
you going to test that hypothesis? Make sure you have appropriate negative controls.
DATA AND OBSERVATIONS
The data will be given to you during the laboratory session. After discussing the procedures that
were developed, and the data distributed, we will discuss what to do with the data.
To hand in:
The report will be due on Thursday August 3rd. You will hand in the predictions and answered
questions from Part 1. You will also hand in the outline of the procedure that you developed for
Part 2. Lastly, you will hand in the data analysis from Part 2, including a graph of the results and
your conclusions.