introduction to genetic engineering bacterial transformation with...

Introduction to Genetic Engineering

Bacterial Transformation with Green Fluorescent Protein (pGLO):

(Inducible Expression)

Teacher Guide

Table of Contents Teacher Guide

NGSS Alignment ........................................................................................................................ T1 Unit Overview & Student Background Needed........................................................................... T4 Inventory Sheet & Reagents & Supplies provided by BABEC ....................................................... T5 Equipment & Supplies Needed at School Site ............................................................................ T6 Student Workstations ............................................................................................................... T7 Suggestion Transformation Laboratory Preparation Sequence ................................................... T8 Teacher Preparation Instructions: Streaking, Aliquoting ............................................................. T9 Important Laboratory Concepts .............................................................................................. T11

Student Guide Introduction to Genetic Engineering ............................................................................................ 1 Background Information and Scientific Theory............................................................................. 2 General Lab Skills Required for Success ....................................................................................... 4 Laboratory Activity ...................................................................................................................... 5 Worksheet: Pre-Laboratory Activity & Student Learning Outcomes ............................................ Q1 Worksheet: Post Lab Questions ................................................................................................. Q3 Worksheet: Calculating Transformation Efficiency ..................................................................... Q6

www.babec.org

1 pGLO Bacterial Transformation

Dec 2017

NGSS Alignment

Disciplinary Core Ideas Components that align to the transformation curriculum?

LS1: From Molecules to Organisms: Structures and Processes

LS1.A Structure and function

All cells contain genetic information in the form of DNA molecules. Genes are regions in the DNA that contain the instructions that code for the formation of proteins, which carry out most of the work of cells.

LS1.B Growth and development of organisms

Cellular division and differentiation produce and maintain a complex organism

LS1.C Organization for matter and energy flow in organisms

As matter and energy flow through different organizational levels of living systems, chemical elements are recombined in different ways to form different products.

LS2: Ecosystems: Interactions, Energy, and Dynamics LS2.An Interdependent relationships in ecosystems

Organisms would have the capacity to produce populations of great size were it not for the fact that environments and resources are finite.

LS2.B Cycles of matter and energy transfer in ecosystems

Bacteria/algae form the lowest level of the food web. At each link upward, only a small fraction of the matter consumed at the lower level is transferred upward, to produce growth and release energy in cellular respiration at the higher level.

LS2.C Ecosystem dynamics, functioning, and resilience

anthropogenic changes (induced by human activity) in the environment—including habitat destruction, pollution, introduction of invasive species, overexploitation, and climate change—can disrupt an ecosystem and threaten the survival of some species.

LS2.D Social interactions and group behavior

Group behavior has evolved because membership can increase the chances of survival for individuals and their genetic relatives

LS3: Heredity: Inheritance and Variation of Traits

LS3.A Inheritance of traits

The instructions for forming species’ characteristics are carried in DNA. All cells in an organism have the same genetic content, but the genes used (expressed) by the cell may be regulated in different ways.

LS3.B Variation of traits Environmental factors also affect expression of traits, and hence affect the probability of occurrences of traits in a population. The variation and distribution of traits observed depends on both genetic and environmental factors.

LS4: Biological Evolution: Unity and Diversity LS4.A Evidence of common ancestry and diversity

Genetic information provides evidence of evolution. DNA sequences vary among species, but there are many overlaps; in fact, the ongoing branching that produces multiple lines of descent can be inferred by comparing the DNA sequences of different organisms.

LS4.B Natural selection

Natural selection occurs only if there is both (1) variation in the genetic information between organisms in a population and (2) variation in the expression of that genetic information—that is, trait variation—that leads to differences in performance among individuals. The traits that positively affect survival are more likely to be reproduced, and thus are more common in the population

LS4.C Adaptation

Adaptation also means that the distribution of traits in a population can change when conditions change. Changes in the physical environment, whether naturally occurring or human induced, have thus contributed to the expansion of some species, the emergence of new distinct species as populations diverge under different conditions, and the decline–and sometimes the extinction–of some species.

LS4.D Biodiversity and humans

Biodiversity is increased by the formation of new species (speciation) and decreased by the loss of species (extinction). Human activity is adversely impacting biodiversity.


Dec 2017

Science & Engineering Practices How does the transformation curriculum align?

1. Asking Questions and Defining Problems

Testable questions on how the phenotype of an organism can be manipulated and changed. Engagement with a phenomenon: glowing organisms. I notice… I wonder….

2. Developing and Using Models

Opportunities for hands-on, iterative modeling of: cell membrane permeability, charge interactions, recombinant DNA technology and genetic engineering

3. Planning and Carrying Out Investigations

Clear procedure and investigation using controls and collection of the appropriate data to allow for revision.

4. Analyzing and Interpreting Data

Numerous data outcomes to analyze and discuss: transformation efficiency, effect of time & temperature, ratios of different colored transformants, role of positive and negative controls, comparison of results between groups and classes.

5. Using Mathematics and Computational Thinking

Transformation efficiency, and calculation of expected result based upon various inputs such as: # of cells, amount of arabinose, time, incubation temperature, etc.

6. Constructing Explanations and Designing Solutions

Applications of transformation in real world and understanding of current issues such as antibiotic resistance. Brainstorming and designing new ways to insert DNA into a cell to solve medical, agricultural and environmental problems.

7. Engaging in Argument from Evidence

Claims on phenotypic change in bacteria (i.e. I changed the DNA), providing evidence from the lab that supports the claim (i.e. it glows), and reasoning on how transformation accounts for the phenotypic change. (i.e. DNA provides ability to glow. i.e.). Argumentation on both sides of an ethical/societal issue regarding antibiotic resistance, GMO’s, insulin production, etc.

8. Obtaining, Evaluating, and Communicating Information

Communicate of basic scientific concepts on DNA structure & function, genetic engineering technology, standard laboratory techniques, and ethical considerations.


Dec 2017

Cross Cutting Concepts How does the transformation curriculum align?

1. Patterns Micro: Formation of bacterial colonies (location & space), growth on plates with and without ampicillin. Macro: global bacterial resistance

2. Cause and Effect 1) link the phenotypic change of bacteria to transformation, and 2) explain why bacteria grow and/or glow under certain experimental conditions.

3. Scale, Proportion, and Quantity

Size and quantity relationship between individual cells and colonies of bacterial cells, and can see the relationship between altering the amount of cells, amount of plasmid, temperature, and time.

4. Systems and System Models Bacterial transformation as a model for the systems of gene expression, and for the manipulation of systems (i.e. interacting cell organelles; biochemical interactions) that allow for genetic engineering.

5. Energy and Matter: Flows, Cycles, and Conservation

Addition of energy into a system (the heat-shock step) and manipulating ionic charge to alter matter (the cell membrane)

6. Structure and Function Genetic modification of the genome (DNA) can induce change in phenotypes (function)

7. Stability and Change

Different environmental conditions such as antibiotic resistance alter living organisms. Cell competency is the ability to take up extracellular DNA from its environment during the process of transformation (heat and charge destabilize the cell membrane).


Dec 2017

Unit Overview One of the biggest challenges of molecular biology is that many of the events and processes studied can’t be seen by the human eye. A great way to provide an enlightening learning experience for students is to use the gene from a bioluminescent jellyfish, Aequorea victoria, that makes green fluorescent protein (GFP). GFP fluoresces a brilliant, glowing green when viewed under UV light and is easily observable. BABEC’s version of green fluorescent protein is the plasmid called pGLO. Using pGLO will allow you to do extension exercises like fluorescent protein purification with affinity chromatography and SDS-PAGE analysis. Bacterial transformation occurs when a cell is given a new piece of genetic material (DNA). This new genetic information often provides the organism with a new trait. These traits can be observable, as in the case of GFP. Bacterial transformation is also called Genetic Engineering, or Recombinant DNA Technology, the revolutionary discovery that launched the biotechnology industry. The technique is especially useful because specific genes can be excised from human, animal, or plant DNA and inserted into bacteria or other cells, to give them new functions. For example, the DNA sequence for the human hormone insulin can be put into bacteria. Under the right conditions, these bacteria can then make human insulin and patients with diabetes can be treated with this “recombinant” insulin. This process has increased the quality and accessibility of treatment for diabetes patients worldwide. Bacterial transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or drought resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest the oil from oil spills. In medicine, gene therapy on humans, or genetically transforming a person with a disease caused by defective genes has not been as successful. Studies to reverse blindness and bleeding disorders have been promising but still require more research.

Student Background Knowledge Needed: To gain the most from this activity, students should already be familiar with:

• the relationship between genes and proteins • basic bacterial parts and their function • aseptic technique and micropipette or Pasteur pipette use • the purpose of antibiotics and their uses.

Questions? Please contact us at: [email protected]


Dec 2017

Inventory Sheet

Listed on the following table are the reagents and consumable supplies provided in the pGLO Bacterial Transformation Kit from BABEC. If you need access to additional materials, please contact us. For this lab, it is a best to work in groups of 4 students. Each group will perform the experiment with +DNA and the negative -DNA samples. The Transformation Kit supports up to 10 groups or 40 students with some overage for aliquoting. pGLO Transformation Reagents & Supplies provided in the BABEC Kit:

✔ Item Storage Amount Per Kit Amount Per Group

Live E. coli culture plate (HB101) Refrigerator (4C) 1 plate a small scrape or smear

pGLO::HB101 plate – for demo only Fluoresces under UV Refrigerator (4C) 1 plate None for students-

use for demo only

10 ng/μl pGLO plasmid, 150uL/tube Freezer (-20C) 1 tube

10 μL

50mM CaCl2 (Transformation Solution) Room Temp (20–25C) 10 mL ~750 μL

LB nutrient broth, sterilized Room Temp (20–25C) 10 mL ~750 μL

Other Equipment/Consumables that are Needed

✔ Item Storage Amount Per Kit Amount Per Group

Inoculation loops, 10 µL size (Or P20 Micropipette with tips)

Room Temp (20–25C) 30 2

Transfer Pipettes: 300µl graduated (Or P1000 Micropipette with tips)

Room Temp (20–25C) 50 4

Transfer Pipettes: 1mL (Or P1000 Micropipette with tips) Room Temp (20–25C) 5

For aliquoting CaCl2 and liquid LB to microfuge

tubes

Poured LB agar plates See Appendix for instructions

Refrigerator (4C) Store “upside down” with lid

on the bottom

20 plates + extra 2

Poured LB agar/Ampicillin plates See Appendix for instructions


on the bottom 20 plates 2

Poured LB agar/Ampicillin/Arabinose plates See Appendix for instructions


on the bottom 20 plates 1

NOTE: If you do not use the frozen reagents by the end of the school year, please contact BABEC to determine if they should be discarded. You will have some left over after each reagent aliquot. Please keep this as stock for emergency needs in the class.


Dec 2017

pGLO Transformation Equipment & Supplies AT SCHOOL SITE:

Item - Equipment Comments

Incubator oven

To grow E. coli cells at 37C overnight. (alternatively, grow on benchtop at room temperature for 2-3 days or top of a warm area)

Thermometer that can read 42C For making sure the water bath is at the right temperature.

Water bath Set to 42C for heat shock step.

Micropipettes (P20, P200, P1000) with tips Necessary if you are NOT using transfer pipettes and loops

Item – Consumables Comments

1.5mL microtubes Clear or color-coded tubes for LB, CaCl2, +/- DNA Need 4 tubes per group.

Disposable gloves Optional

Item – Other Comments

Microtube racks For holding the tubes

Crushed ice For chilling E. coli cells before heat shock.

Styrofoam cups For holding crushed ice at work stations.

Permanent markers For marking microfuge tubes, plates, etc.

Distilled water For filling water bath; prevents contamination.

Foam microtube holder/float To keep tubes floating in the water bath

Timer To keep track of the heat shock time.

Waste containers or beakers For disposal of pipettes, loops, tubes, etc.

Disinfectant 10% bleach; to disinfect any surface that contacted the bacteria.

UV light source Transilluminator or hand-held black light lamp (optional: transformed bacteria is neon green under UV but light beige under visible light)

Safety goggles To protect eyes when looking into UV light


Dec 2017

Student workstations – How many students does this kit allow?

It is recommended that students work in groups of four so they have both the experimental sample (+DNA) and the negative control (-DNA). This kit serves up to 10 student groups of 4 students each or 40 students total. See the figure below for each student group set up: For each group, at the start of the class, have ready: Have on hand, ready to distribute to each group:

1 Cup of ice 1 Microfuge rack 4 transfer pipettes

Or P1000 & tips 1 Permanent Marker

4 tubes: 1 CaCl2; 1 LB, 2 blank tubes students label -DNA and +DNA and their “team” name

2 loops (teacher distributes so no ends are touched)

5 plates total – 2-LB agar, 2-LB /Amp, 1-LB/Amp/Ara

CaCl2

LB + DNA

- DNA

or

DNA


Dec 2017

Suggested Transformation Lab Preparation Sequence

Activity When Prep Time *

1 ¨ Read this packet, create a lesson plan.

¨ Make copies of the Student Guide and worksheets.

Before starting lab prep

2 hours or more

2 ¨ Streak starter plates using an inoculation loop and the E.

coli culture plate. ¨ Alternatively, have students make their own team starter

plates from the E. coli culture plate ¨ Aliquot ~750μL of LB broth and ~750μL of CaCl2 for each

team into microfuge tubes (color coded if possible).

24-36 hr prior to student use

30 min or more

3 ¨ Set-up student group stations. ¨ Fill hot water bath with distilled water and set to 42C. ¨ Obtain crushed ice and store in nearby freezer

Night before or prior to start of lab

1 hour

* Time needed depends on number of group stations and class sections.

Outline for Class Implementation (suggestions only, and optional) Day Activity

1 Engage students with a phenomenon. Explain transformation. Have students draw a flowchart of the lab activity as homework, before performing the lab activity.

2 Explore by practicing using transfer pipettes with water in a cup and/or streaking starter plates 24 hours before the start of the experiment. Explain colonies vs lawn morphology of bacterial growth on plates. Assign the pre-lab worksheet: make a prediction about the types of plates you need for each transformation –DNA or +DNA; make a prediction of the expected results.

3 Explore with basic lab activity - Transformation and streaking plates.

4 Elaborate with transformation data collection and analysis

5 Elaborate with an extension activity: Determining transformation efficiency or different conditions of

transformation

6 Optional: Elaborate with extension activity: Student group presentations, Bioethics, uses of genetic engineering in science

6 Evaluate and assess student understanding


Dec 2017

Teacher Laboratory Preparation Instructions Streaking starter plates of E. coli Starter plates are needed to produce bacterial colonies of E. coli on agar plates. LB agar plates should be streaked to produce single colonies and incubated at 37C for 24–36 hours before the transformation investigation begins. Under favorable conditions, one cell multiplies to become millions of genetically identical cells in just 24 hours. There will be millions of individual bacteria in a single millimeter of a bacterial colony. Depending on time, you may prefer your students to learn how to streak their own plates for individual colonies. Plate Streaking Streaking takes place sequentially in four sections. The first streak spreads out the cells. In subsequent streaks the cells become more and more dilute, thus increasing the likelihood of producing single colonies.

1. Draw quadrants on the underside of the petri dish. Using a sterile inoculation loop or sterile pipette tip, pick up one bacterial colony from live E. coli culture plate.

2. Using a back and forth motion, gently spread the colony into one quadrant of the LB starter plate. Keep the lid slightly tilted open - only as much as necessary. Be careful not to puncture the agar.

3. Rotate the plate one-quarter of a turn. Go into the previous streak about two times and then back and forth as shown for a total of about 5-10 times.

4. Again, rotate the plate one-quarter of a turn and pass over a previous streak from the previous quadrant several times with the loop.

5. Repeat step 3, but this time, drag out the loop to form a tail not touching any previous streaks. Close your plate to avoid further contamination.

6. Place the used loop (or tip) in a disinfectant solution waste cup. Follow this procedure for the remaining starter plates. Once starter plates are inoculated, incubate them upside down (lid on the bottom) in a 37C incubator oven for 24 to 36 hrs.

7. If your students are not using the plates right away, seal the sides with Parafilm or lab tape so they don’t dry out, invert the plates, and place them in a dark cupboard until needed. Avoid refrigerating your starter plates as cooling will reduce your transformation efficiency.

1 2

34


Dec 2017

What to expect the next day You should see individual bacterial colonies in quadrant 4, and very dense bacterial growth in quadrant 1. Quadrants 2 and 3 will have bacterial density somewhere in between, similar to what is seen below:

Your streaked plate should look similar to this image after 24 – 36 hours:

Note: the images on this page have been provided by the Florida Institute of Technology

Aliquoting LB Broth and CaCl2 (Transformation Solution) Do this quickly and as aseptically as possible.

1. Ready: • Ten - 1.5mL microfuge tubes on a microfuge tube rack. (You might want to use different colored microfuge

tubes to further differentiate these solutions.) 2. Using the 1mL transfer pipette or P1000 Micropipette, aliquot ~750µL of LB broth into each tube. 3. Close the tubes, label the lid with LB. 4. Repeat steps #1-3, this time labeling and aliquoting ~750µL with CaCl2. 5. Leave at room temperature or refrigerate aliquots until needed.


Dec 2017

Important Laboratory Concepts

Lab Safety and Disposal If you do not have an autoclave readily available, all solutions and equipment coming into contact with the bacteria such as the pipette tips and inoculating loops, should be collected and placed into disinfectant solution, such as 10% bleach. Cover each contaminated petri dish with disinfectant solution and let sit for at least 15min before disposal according to your site guidelines. For your protection, wear safety glasses and a lab coat when handling concentrated and dilute disinfectant. Make sure there is adequate ventilation. Your school’s specific safety and disposal policies should always take precedence.

The E.coli used in this lab E.coli is a bacteria found everywhere in our environment. The strain we use for this lab and in many research scientific labs are harmless to humans and are NOT pathogenic. They have been specially engineered to help scientist with their work. If you touch the bacteria with your hands, simply wash with soap and water. If you get some bacteria in your eyes, simply flush with water. As always, use safety precautions when working in the laboratory.

Media and Additives LB (Luria-Bertani) agar and broth contain a yeast extract with a mixture of amino acids, carbohydrates, salts and vitamins. Together, these substances support bacterial growth. Agar contains a gel derived from seaweed that solidifies at room temperature. If you have extra prepared dishes, allow your students to touch the surface of one of the LB plates and help them make connections between agar and Jello. Including the antibiotic ampicillin in the media prevents the growth of bacteria other than the successful transformants. The pGLO plasmid contains a beta-lactamase gene, which allows the transformed bacteria to produce an ampicillin inactivating protein, the enzyme beta-lactamase. Using ampicillin in the media insures that only bacterium containing and producing beta-lactamase will grow. Seeking survival of only the transformed cells is an example of antibiotic selection. Ampicillin breaks down over time and is sensitive to heat and repeated freeze/thaw cycles. Transcription of the pGLO gene then translation of the transcript results in the expression of the GFP protein. The GFP protein allows the transformed cells to appear neon green with a long-wave UV lamp or standard UV transilluminator. The phenotypic expression of the “wild-type” bacteria is white. Transformed cells will also appear green under visible light and fluoresce green under UV light. This engineered pGLO plasmid allows students and teachers to easily verify their transformation success.

Transformation Solution, 50mM CaCl2 When fully intact, the bacterial cell membrane does not allow DNA to pass through it, so how do we get the DNA inside during transformation? We add Ca2+ cations, which neutralize the negative charges of both the DNA phosphate-backbone and the phospholipids within the cell membrane. By neutralizing these repulsive negative charges, the DNA can then easily pass across the bacterial cell membrane. It is possible to get transformants if CaCl2 is missing. However, the efficiency (number of colonies on plates) might be very low. A great animation of the process is available at http://www.dnai.org > Manipulation > Techniques > Transferring & Storing > Transformation Animation.

Heat Shock Heat shock helps bacterial cells take in small foreign DNA segments such as plasmids by increasing a cell membrane’s permeability. Students must carefully follow the pre-optimized process laid out in this protocol; it contains specific temperatures and incubation times that will ensure success. Otherwise, few, if any, bacteria will uptake the plasmid and be transformed.


Dec 2017

Recovery

The 10 min incubation period in nutrient LB broth after the stress of heat shock allows the transformed bacteria cells to heal and grow. They will also begin to secrete beta-lactamase, the ampicillin inactivation enzyme, which increases the survival rates of the transformed cells on the ampicillin plates.

Incubation Optimal growth for E.coli occurs at 37C. E. coli will require more time to grow and express the GFP gene if kept at room temperature. A warm spot on top of the refrigerator or heating units in the classroom will help. It will take 2-3 times longer for the bacteria to grow at room temperature versus an incubator set at 37C, but they will grow in these conditions.

Antibiotic Selection The pGLO plasmid, which contains the GFP gene, also contains the gene for beta-lactamase. Beta-lactamase is an enzyme that provides resistance to the antibiotic ampicillin, a member of the penicillin family. The beta-lactamase protein is produced and secreted by bacteria that contain the plasmid. Beta-lactamase inactivates the ampicillin present in the LB nutrient agar to allow bacterial growth. Only transformed bacteria that contain the plasmid and express beta-lactamase can grow on plates that contain ampicillin. Only a very small percentage of the cells successfully take up the plasmid DNA during heat shock and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. In order to "stably retain" the plasmid, there needs to be some type of metabolic reason for the E. coli to keep the plasmid around. If the plasmid contains a gene that codes for a protein that protects against antibiotics, then only cells that have the plasmid will survive in the presence of that antibiotic

Aseptic technique When growing bacteria in culture, it is important to prevent the growth of unwanted microorganisms in the nutrient rich media. Aseptic technique is a series of methods that are used to minimize the chances of contamination. Examples include use of sterile tubes and pipettes, sterilized solutions, cleaning the work area with disinfectants, use of Bunsen burners, and keeping the caps of tubes, plates and pipette boxes closed.


Dec 2017

Introduction to Genetic Engineering: Bacterial Transformation with Green Fluorescent Protein (pGLO)

Genetic engineering is an umbrella term that encompasses many different techniques for moving DNA between different organisms. Transformation is the process by which an organism acquires and expresses a whole new gene. In this activity, you will have the opportunity to genetically transform bacteria cells; altering them so that they can make an entirely new protein. This procedure is used widely in biotechnology laboratories and has enabled scientists to manipulate and study genes and proteins in exciting new ways. In this procedure, students will add a gene that codes for Green Fluorescent Protein (GFP). This protein was discovered in the bioluminescent jellyfish called Aequorea victoria, a jellyfish that fluoresces and glows in the dark (Figure 1). The gene for GFP was isolated in 1994 and was quickly used in laboratories as a way to brightly label proteins in a living cell. This “tagging” of proteins allowed researchers to visualize specific proteins to learn more about their biological functions in exciting new ways. The discovery of GFP proved to be so important that the Nobel Prize in Chemistry was awarded to Osamu Shimomura, Marty Chalfie and Roger Tsien in 2008 for their work. Since then, Roger Tsien’s laboratory at UCSD has altered the GFP gene to make a full rainbow of proteins. Figure 2 shows how bacterial expressing many different colored fluorescent proteins can be grown together on one plate.

Bacteria are commonly used for genetic transformation experiments because they are simple, single-celled organisms that grow and reproduce very quickly. Bacteria cells store their DNA on one large, circular chromosome. But they may also contain one or more small circular pieces of DNA called plasmids. Because bacteria reproduce asexually, plasmid DNA allows for the addition of new traits into a cell. Plasmids replicate independently of the large bacterial chromosome, and can transfer easily between cells. Figure 3 shows the circular DNA chromosome and plasmid DNA inside of a cell. Bacterial evolution and adaptation in the wild often occur via plasmid transfers from one bacterium to another. An example of bacterial adaptation is resistance to antibiotics via the transmission of plasmids. This natural process can be modified by humans to increase our quality of life. In agriculture, genes are added to help plants survive difficult climatic conditions or damage from insects, and to increase their absorption of nutrients. Toxic chemical spills can often be bio-remediated (cleaned-up) by transformed bacteria specifically engineered to do the task. Currently, many people with diabetes rely on insulin made from bacteria transformed with the human insulin gene. Scientists use transformation as a tool to study and manipulate genes all the time.

Figure 1: Aequorea Victoria glowing under UV light Retrieved from: http://voices.nationalgeographic.com/2012/04/03/love-and-war-the-essence-of-luminosity/

Figure 2: A rainbow of fluorescent growing on an agar plate. Retrieved from: http://www.tsienlab.ucsd.edu/Images.htm

Figure 3 Genetic material in bacteria takes 2 forms. Retrieved from: Wikipedia. https://en.wikipedia.org/wiki/Plasmid


Dec 2017

Background Information and Scientific Theory

The Central Dogma of Molecular Biology A basic tenet of biology, from single-celled bacteria to eukaryotes, is the mechanism of coding, reading and expressing genes. The central dogma of molecular biology states that: DNA > RNA > PROTEIN > TRAIT. This curriculum is an example of the central dogma in action. The instructions for GFP production are encoded in the DNA. When transcription is turned on, the cell turns those instructions into an mRNA transcript. This transcript is then translated into protein, which provides the trait of fluorescence.

Gene Regulation Every cell in the human body shares an identical genome that contains over 20,000 different genes. But if all cells have the same genes, how is it that a muscle cell ends up being so very different from a brain cell? The answer lies in the fact that there is a specific process for controlling which genes are turned “on” and which are turned “off” in every single cell. Gene regulation is the name for all the different cellular processes that have to take place in order for a gene to be turned into a protein. Gene regulation is an important concept in biology. Where and when genes are turned on or off, called gene expression, results in the expression of proteins – the workhorses of the cell. Proteins called transcription factors are frequently used by cells to turn transcription on or off depending on environmental conditions. They are important for cellular development, tissue specialization, and organismal adaptation to the environment. Transcription factors act at the promoter region in front of a gene. At the promoter RNA polymerase initiates transcription and turns a gene on; the gene is then said to be “expressed”. Once the mRNA transcript is made, it can be translated into protein. All the genes in our bodies are highly regulated to allow for maximum efficiency, and to decrease waste (energy) in our cells.

The pGLO System In this laboratory activity, you will have the opportunity to genetically engineer a cell and you will see with your own eyes the critical role of gene regulation in living systems. This is because the expression of the GFP gene in this experiment is not automatic. Rather, it happens only when the environmental conditions are just right. Plasmids used by molecular biologists are named with an acronym that begins with the lower case "p", and followed by a name that conveys information about its function. pGLO is the name for a plasmid that has been engineered to contain the gene for GFP, which glows under UV light. Using recombinant DNA technology, scientists designed this plasmid to contain two additional genes, for a total of three genes whose function is important to understand before beginning this activity. In this lab, you will be using non-pathogenic E. coli bacteria and pGLO, a plasmid modified with three genes. The pGLO plasmid contains the genetic codes for (see Figure 4): Figure 4: pGLO plasmid and its three important genes.

Gene: pGLO plasmid with inserted genes

Ampr: Ampicillin resistance gene forming beta-lactamase, which inactivates ampicillin in media

GFP gene: codes for green fluorescent

protein, which is derived from Aequorea Victoria – a bioluminescent jellyfish that fluoresces under UV light.

Ara: codes for the regulatory protein araC which works with the sugar arabinose to turn on GFP transcription by recruiting RNA polymerase to the promoter.

pGLO Plasmid

ampr

ara


Dec 2017

In a process termed bacterial transformation, you will introduce the pGLO plasmid into bacteria. The procedure is never 100% efficient and only a few of your E. coli bacteria will successfully “take up” the pGLO. How will you know which cells contain the plasmid? pGLO contains a gene that codes for a protein that protects the cell against the toxic effects of antibiotics. This means that only cells that have the plasmid will survive in the presence of antibiotics. In this procedure, we use ampicillin, an antibiotic very similar to penicillin. This step is called antibiotic selection, and it allows you to select only the cells that have been transformed. The beta-lactamase gene in pGLO codes for a protein that breaks down ampicillin. Expression of the beta-lactamase gene in cells that have been successfully transformed allows them to grow in the presence of ampicillin. Non-transformed cells will die. Your transformed cells will grow on a plate with ampicillin, but they will not fluoresce green until the GFP gene is turned “on”. Here’s where the idea of gene regulation comes into play. Transformed cells will grow on plates not containing arabinose, but will only fluoresce green under UV light when arabinose is included in the nutrient agar. Therefore, arabinose, a sugar that bacteria consume for energy, is the critical ingredient for making your bacteria glow. What’s so special about arabinose? It teams up with the araC, the regulatory protein that was added to pGLO. Regulatory proteins control the timing and location of many cellular processes. Specifically, araC is a transcription factor which, as described on the previous page, functions to turn genes on and off. But it can’t turn GFP on by itself – it needs the help of arabinose. Together, they work to bring in RNA polymerase, the enzyme that makes RNA, and only then can the glowing, green protein be made. It's a finely orchestrated dance, and all the right players have to be in place for success.

Figure 5 Gene regulation of GFP in pGLO

Figure 5 shows that when araC teams up with arabinose, its shape changes. The protein araC easily forms a bond with the sugar arabinose, and only when they both get close together can the complex function as a transcription factor. It then calls in RNA polymerase to start transcription, and we see firsthand the central dogma of molecular biology in action!

The Transformation Procedure In order to increase the chances that your E. coli will incorporate foreign DNA, you will need to alter their cell membranes to make them more permeable. This is a three-step process.

1. First you place your cells and pGLO together in a transformation solution (which contains calcium chloride) to neutralize the charge.

2. Second, you quickly heat shock them with a temperature change (42oC). This hot temperature permeabilizes (loosens) the bacterial cell wall, making it easier for pGLO to cross it.

3. This process can be harmful to the cells, so you want to give them a nutritious broth to restart their growth as soon as you’re done. Luria Broth (LB) is a liquid that contains proteins, carbohydrates and vitamins so that the E.Coli can rapidly recover and thrive. They will then be placed on an agar medium, a jello-like substance containing LB, with or without antibiotic or sugar, to grow overnight.

If pGLO transformation is successful and the bacteria are growing, the colonies will appear light beige under natural light, neon green under UV light. These green bacteria must contain the plasmid with the GFP. For this reason, the green fluorescent protein (GFP) gene is often used as a “reporter gene” to identify expression of other genes of interest. Note: the “p” in pGLO stands for plasmid while the protein is Green Fluorescent Protein (GFP.)


Dec 2017

General Lab Skills Required for Success

Using Sterile Technique Students should wash their hands before starting lab, after handling recombinant DNA organisms/containers, and before leaving the lab area. All lab surfaces should be decontaminated at least once a day during each class section and following spills. Students should avoid touching the tips of the pipettes or inoculating loops onto any contaminating surfaces, unless instructed in the protocols.

Using Transfer Pipettes A transfer pipette works just like an eye-dropper. Observe the 100µl, 200µl and 300μl marks on the transfer pipette. You will need to transfer a volume of 250μl, found between 200μl and 300μl. You will also need to transfer a volume of 150μl. Where do you think that volume will be found? When using, bring the pipette up to eye level to confirm that liquid has been transferred correctly. For practice, get a feel for the pipette by transferring water from one container to another. Success of the lab depends on the proper use of tools and reagents required for the protocol.

Using Inoculating loops

You can measure precisely 10µl with a 10μL inoculation loop. Dip the loop into the tube containing liquid. A noticeable film will form around the ring due to surface tension (like a bubble wand). Swirl the loop into the tube using your index finger and thumb. Note that the loop may not fit into the narrow bottom end of most microtubes.

UV Safety Ultraviolet radiation can cause damage to eyes and skin. If possible, use UV-rated safety glasses or goggles if looking directly at UV light.

Using Experimental Controls In this lab, it is important to confirm which cells have received the plasmid, and under which conditions the green fluorescent proteins are being produced. You will need to prepare experimental controls to be able to interpret your results correctly. These controls are designed to minimize the effects of factors other than the single concept that you are testing. Therefore, each student group will perform 2 different reactions: one with pGLO plasmid (+DNA) and one without it (- DNA). Each reaction then, will be plated on LB only and LB/AMP plates.


Dec 2017

Name: __________________________

Laboratory Activity

The protocol outlined next describes the procedure for bacterial transformation. Follow the steps very carefully for higher likelihood of success. Make sure you work aseptically and accurately. Reminders:

1. Do not open anything until needed. 2. Do no touch the ends of the instruments. 3. Always work from negative (-) to positive (+). Why? 4. Make good use of your time. While waiting for short incubations, read and get ready for the next step.

Before your start, make sure your group has the following items:

¨ Cup of crushed ice ¨ 1 tube labelled CaCl2 ¨ 1 tube labelled LB ¨ 2 empty/blank tubes ¨ Tubes should be in a rack ¨ 4 transfer pipettes (DO NOT OPEN UNTIL NEEDED) ¨ 1 Permanent marker (may have to share with the whole class)

Place a check mark in the box as you complete each step.

pGLO Transformation Protocol

1. STERILIZE lab surfaces and wash hands before

beginning the lab. (Why?)

2. LABEL TUBES: Using a permanent marker, label one

empty microfuge tube +DNA and the other -DNA. Put your team name on the side of the tube.

Label each tube twice, on the lid and on the side. Place these tubes into a Styrofoam cup containing crushed ice.

3. ADD 250μL of CaCl2 to each tube using a sterile

transfer pipette. Or a micropippette. If using a P200 micropipette, use 125mL twice.

Note: TS contains calcium chloride (CaCl2), which helps neutralize both the bacterial cell wall membrane and DNA charges. Keep your tubes on ice.

250μL CaCl2

or

+ D

NA

− D

NA


Dec 2017

4. PICK UP BACTERIA FROM PLATES: With a sterile

inoculation loop that the teacher gives you (Hold the loop in the middle. DO NOT TOUCH THE ENDS), pick up a small smear of bacteria from the starter plate, using the small end.

Dip and swirl the loop into the -DNA tube to evenly disperse the colony in the solution and release it from the loop. With the cap closed, flick the tube with your finger to mix. Repeat but put the bacteria into the +DNA tube. Return tubes to ice.

5. ADD the pGLO DNA: With the 10 μL inoculation loop,

dip the big loop into the stock plasmid tube. A noticeable film will form around the ring due to surface tension (like a bubble wand). Swirl the loop into tube labelled +DNA only.

Micropipette: Use a p-20 and put 10 μL into the tube. What should the dial window look like?

**DO NOT add plasmid to the –DNA tube.

Close the cap and flick the tube to mix. Quickly, place the tubes on ice.

10 μL pGLO

6. INCUBATE ON ICE: Incubate both tubes on ice for 5

minutes, making sure the tubes are in contact with the ice.

Note: This step allows the charges to neutralize so the cells can take up the plasmid DNA in the next step. While waiting, read through the next step as it is CRUCIAL for success.

5 minutes on ice

7. HEAT SHOCK your bacteria by transferring both tubes

to a foam rack and placing them into a water bath set at 42C for 50 seconds.

Make sure the tubes are pushed down as far as they can go in the rack to contact the hot water. After 50 seconds, quickly place both tubes on ice for another 2 minutes. It is VERY important to watch the time and speed of the transfers.

+ D

NA

+ DN

A

− DN

A

+ DN

A

− DN

A

Water&Bath&42°C%/%50%seconds%

+ DN

A

− DN

A

2%min%

+ D

NA

− D

NA


Dec 2017

8. FEED THE CELLS WITH FOOD:

Return your tubes to a tube rack. Using a sterile transfer pipette, add 250 μL of LB broth to each of the tubes. Close the tubes. Mix each tube by flicking it several times with your finger.

Or use a micropippette, If using a P200, use 125 μL twice. What should the dial window look like?

250μL LB

9. LET THE CELLS RECOVER:

Incubate the tubes for 5-20 minutes at 37C. You can use the bacterial incubator or other warm place like the top of a refrigerator for this step.

Note: This process allows the transformed bacteria to recover from the “shock” by providing nutrients for their growth.

- DNA + DNA

5-20 minutes at 37°C

10. LABEL ALL PLATES: While you’re waiting, pick up: 2-LB

and 2-LB/amp and 1-LB/amp/ara plates. Why these plates for the type of transformations? On the bottom of the plate (non-lid side and the edge of the plates), write the date, your initials or your team initials, and - DNA or + DNA onto the LB and the LB/amp plates. Use the picture on the right to guide you.

11. PLATE CELLS. Using a transfer pipette, transfer 150μL

of the -DNA to each plate labeled -DNA. Slowly pipette/dribble directly onto the agar (bottom plate with agar, not the lid!) Allow liquid to soak into agar.

Then do the same for the +DNA, using the same transfer pipette. By going from -DNA to +DNA, you are using good lab practices and saving on resources. Swirl to coat the agar evenly. Allow bacteria to saturate (and dry) into the agar plate for a few minutes before the next step.

- DNA + DNA

or


Dec 2017

12. INVERT, TAPE, INCUBATE. Once the liquid is soaked

up, invert your plates (so lid is again on the bottom). Then stack and tape them together.

Place plates into an incubator oven set at 37C until the next day or when colonies are visible. Alternatively, stack the plates into a warm spot in the classroom. It may take 2-3 days for bacterial colonies to appear. After the colonies have appeared, you can observe and analyze your results! Take pictures! For storage, you may keep the plates by wrapping them in parafilm or tape and storing them in the refrigerator.

14. CLEAN UP, WASH UP: When done with all lab activity, clean up you trash and instruments. Decontaminate all lab surfaces with dilute disinfectant and wash hands!

10% Bleach

Name:_______________Period______

Date:___________________________

Q1 pGLO Bacterial Transformation

Dec 2017

Pre-Lab Activity: Student Learning Outcome & Pre-Lab Predictions For Laboratory Activity

Learning Outcomes:

• Explain the process of bacterial transformation. • Relate the use of bacterial transformation in biotechnology. • Differentiate transformed from non-transformed cells. • Calculate transformation efficiency and compare with class data.

1. What plates do you need? In order to determine if your bacterial transformation worked, what plates do you need to

“streak” your transformed bacteria? Remember, you have – DNA bacteria and + DNA bacteria. You have three choices of plates, LB agar, LB/amp and LB/amp/ara. Draw a picture or a table to determine what plates you need for each type of bacteria and explain why. Why do you use a – DNA control?

2. Explain the purpose of these processes or substances during transformation.

Process or Substance Purpose

a. LB agar

b. Ampicillin or antibiotic

c. Calcium chloride

d. Heat shock

Name:_______________Period______

Date:___________________________


Dec 2017

3. Predict the results by filling in the Pre-Lab before you see the results of the experiment. Lab Predictions: Answer the lab prediction questions below by filling in the table. Make your prediction and fill out the table below:

Item Prediction growth on LB

Prediction: growth on LB/amp

Prediction: growth on

LB/amp/ara

Will it be green?

Explanation of Your

Prediction

Untransformed bacteria ( - DNA tube)

Transformed bacteria ( + DNA tube)

Plasmid only (DNA only)

Name:_______________Period______

Date:___________________________


Dec 2017

Post-Lab Questions

1. Post-Lab Results. Put your results here:

- DNA on LB

- DNA on LB/Amp

+ DNA on LB

+ DNA on LB/amp

+DNA on LB/amp/ara

Illustration of Results

Description of results

2. Compare your predictions with your actual lab results. Describe how close your predictions were to your actual results and explain possible reasons for any differences. Was the transformation protocol successful in creating ampicillin resistant? Give evidence and reasoning on your claim.

Name:_______________Period______

Date:___________________________


Dec 2017

3. Describe 2 differences and 2 similarities between these bacteria.

Condition - pGLO DNA bacteria + pGLO DNA bacteria

Difference

Similarity

4. Explain what may have occurred to produce these results. ( • = colony)

Contents LB -DNA LB/amp -DNA

Illustration of Results

Description of Results

Possible explanation for results

Name:_______________Period______

Date:___________________________


Dec 2017

5. If growth appeared on the LB/amp +DNA plate, would these bacteria be transformed? Explain.

Sometimes, smaller nontransformed satellite colonies are seen around a central transformed colony. Are they any satellite colonies on your plates? Record the satellite colonies’ morphology (size, shape, and color) compared with the transformed colonies and reasons for any difference.

6. Provide an example of how transformation can be beneficial and an example of how it can be potentially harmful to humans. You may look up information on the web.

Condition Transformation example

Beneficial

Harmful

Name:_______________Period______

Date:___________________________


Dec 2017

Worksheet: Calculating Transformation Efficiency When performing transformation experiments, you usually want to obtain as many transformants as possible. This is important because you want to make sure your conditions for transformation is at its optimum. Transformation efficiency is the efficiency whereby cells take up the introduced DNA. Many factors contribute to transformation efficiency: cell age and competency (the ability to take up DNA), the type of cells being transformed, plasmid length and quality, the method of transformation (heat shock or electroporation) and just different conditions in general. Having a low transformation efficiency may point to poorly competent cells, poor conditions, or poor techniques (not following protocol). In a research lab, it’s good to have many transformants for research, just in case individual transformants may not work as well (e.g. different levels of expression), or some other unknown problems associated with transformed cells. In making a genomic library, you want as many transformants as possible to have a robust library. In cell culture, you may take a population of transformed cells for further study therefore having a high transformation efficiency allows for better study. In this exercise, we will calculate the transformation efficiency of the E. coli bacteria by pGLO . The data can then be gathered from each team of the class and the data compared with a different transformation technique called electroporation. Two data are needed for this:

1. Total number of green fluorescent colonies on your LB/amp plate. 2. Total amount of pGLO plasmid DNA used for bacterial transformation that was spread on the LB/amp plate.

1. Determine the total number of transformed green fluorescent colonies. Place the LB/amp plate near a UV light source. Count the number of green fluorescent colonies that glow under UV light. Enter that number here à 2. Determine the amount of pGLO DNA in the cells spread on the LB/amp/ara plate.

a. Total amount of DNA: DNA in μg = (concentration of DNA in μg/μL) x (volume of DNA in μL) In this experiment, 10μl of pGLO at a concentration of 0.01μg/μL was used. Enter that number here à

Total number of colonies = _____________

Transformation efficiency calculation: The number of colonies observed growing on an agar plate (cfu) Amount of DNA used (in μg) cfu=colony forming units

Total amount of pGLO DNA, µg used in this experiment = _____________

Name:_______________Period______

Date:___________________________


Dec 2017

b. Fraction of pGLO plasmid DNA (in the bacteria). For this experiment, a certain amount was spread onto each plate. To find that fraction:

Fraction of DNA used à

• 150μl of cells was spread from the tube containing a total volume of 500μl of solution.

Enter that number here à

c. How many μg of pGLO DNA was spread on the LB/amp/ara plate? Multiply the total amount of pGLO DNA used by the fraction of pGLO DNA you spread on the LB/amp/ara plate.

pGLO DNA spread (μg) = amount of DNA used (μg)(a) x fraction of DNA(b) Enter that number here à Now, we are finally ready to calculate the transformation efficiency! Enter that number here à

Sample volume spread on LB/amp/ara plate, in µl Total sample volume in tube, in µl

Fraction of DNA= _____________

pGLO DNA spread, µg = _____________

Number of colonies on LB/amp plate = _______________ pGLO DNA spread, µg = _____________

Transformation efficiency calculation: The number of colonies observed growing on an agar plate Amount of DNA used (in µg)

Transformation efficiency = _____________ transformants or cfu/µg cfu=colony forming units

Name:_______________Period______

Date:___________________________


Dec 2017

1. Analysis of results: What is the transformation efficiency of each team in the class?

Team Efficiency

a. Calculate the Mean, Median and Mode of the results from above.

i. What was the average transformation efficiency?

ii. What was the Median?

iii. Was there a Mode?

Name:_______________Period______

Date:___________________________


Dec 2017

2. In past studies, this method of “heat shock” protocol that was performed by research labs usually has a transformation efficiency between 8x102 and 7x103 transformants per microgram of DNA.

a. How does your team’s result compare to this data?

b. How does the class’ result compare to your data and to the data by research labs? 3. Not all the cells in the culture are transformed. What evidence do you have to support this statement? 4. Another method for transformation is called electroporation. In this method, an electric field is applied to allow the cell

membrane to open up and take up DNA. The transformation efficiency from electroporation may be 1x108 cfu/μg.

a. What fold higher is the transformation efficiency by electroporation vs. heat shock?

b. How does electroporation compare to the heat shock method?

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