DNA is the programming within a cell.

Cells can be reprogrammed by humans to act as sensors or to complete more complex tasks.

Plasmids are small DNA messages that can be used by bacteria to alter their programming and behavior.

Antibiotics can be used as a way to select for genetically engineered bacteria.

Genetic Engineering

Plasmid

Transformation

Ampicillin

Arabinose

pGLO

LacZ

GFP

• pBLU plasmid (LacZ) (Carolina, item # 211427)
• extra e.coli (Biorad, item # 1660408EDU)
• Luria Broth Agar + Ampicillin plates (Carolina, item # 216604)
• Luria Broth Agar + Ampicillin + xGal plates (Carolina, item # 216604)
• pGLO Bacterial Transformation Kit (BioRad, item # 1660003EDU) contains:
• Transformation competent E. coli bacteria
• pGLO Plasmid DNA
• Agar plates with or without antibiotic and arabinose sugar
• Transformation solution
• Nutrient broth
• Disposable bulb pipettes
• Inoculation loops/spreaders
• UV light
• Instruction manual with detailed instructions on how to set up this lab
• Extra UV lights (E737)
• Micropipettes 200 ul, optional (E642)
• Micropipettes 20 ul, optional (E641)
• Ice and containers for ice
• 40 deg C water bath (or beaker of water on a hot plate set at 40 deg C)
• 37 deg C incubator (if not incubator available, cultures can be grown at room temp for 2 days, instead of 24 hours, or use an electric blanket to slightly warm the cultures and speed up growth

Daly Ralson Resource Center:

Blacklights (E737)

Micropipettes 200 ul (E642)

Micropipettes 20 ul (E641)

Groups of 3-4 students

3 days

Day 1

5 min – Intro

20 – Set up biosensors

Day 2

20 min – Observe biosensors

20 min – Intro to transformation

60 min – Bacterial transformation

20 min – Experiment Setup

Day 3

20 min – Observe bacterial transformation

30 min – Wrap-up discussion

An understanding of the basics of cell biology is helpful, particularly the central dogma (DNA à RNA à Protein), and cell mitosis. If you would like to perform this activity using micropipettes, basic understanding of pipetting techniques is helpful. However, the instructions for this kit are written assuming students are using disposable bulb pipettes.

• Assess students’ prior knowledge and conceptions about genetic engineering.
• Reinforce DNA as the cell’s program/instructions. Develop student’s understanding that by altering the cell’s genes, the cells gain new functions and behaviors.
• Introduce the concept of a biosensor, a cell that acts as a living sensor.
• Develop students’ understandings of how both the genes (program) and the environment affect cell behavior.
• Introduce gene expression and regulation as programming blocks

Biosensors are devices that combine a biological component with a physiochemical component to report the presence of a chemical using a sensitive living sensor. The biological component, in this case, is e. coli bacteria. By adding plasmid DNA to the e. coli bacteria, we are converting them into a biosensor that can create an optical output, the physiochemical component, that reports the presence of a chemical. A very simple example of a biosensor is a canary in a coal mine. The canary would die if certain toxic substances were in the air, but at significantly lower concentrations than would be deadly to humans. Many cellular biosensors are similar, used to detect toxic substances at concentrations far lower than humans can detect. (see: https://www.wired.com/story/this-digital-pill-prototype-uses-bacteria-to-sense-stomach-bleeding/, https://www.eurekalert.org/pub_releases/2019-05/ru-sbh052019.php)

The LacZ gene is part of the lac operon, a bit of DNA required for the transport and metabolism of lactose in e. coli.  The lac operon consists of structural genes, and a promoter, a terminator, regulator, and an operator. LacZ is one of the structural genes and encodes for β-galactosidase, an enzyme that cleaves the disaccharide lactose into glucose and galactose.  Scientists use their knowledge of this enzyme to create a reporter system. X-gal, which is a compound consisting of a galactose linked to indole. When LacZ (β-galactosidase) cleaves X-gal, indole forms a compound similar to indigo dye and the bacteria turn blue.

pGLO is an engineered DNA plasmid vector. It has several parts, including a gene switch, ampicillin resistance, and the GFP gene. The gene for GFP was originally isolated from the jellyfish, Aequorea Victoria, and under UV light, will glow green. Therefore, when the bacteria are transfected with pGLO, they have DNA from two different species, making them a chimera. The plasmid’s gene switch is activitated by the sugar arabinose.  So, although bacteria may have the pGLO plasmid, they will only glow in the presence of arabinose (and under UV light).

The BioRad bacterial transformation kit comes with detailed instructions on how to prepare for the activity.  Please note, that our activity uses only half of the poured plates, and only uses three plates per group, instead of four (only one LB + Amp plate). We find that this is less confusing for student than the original BioRad setup.

If you plan to do the LacZ biosensor activity, you will need to use a portion of your BioRad kit, along with the extra e. coli. The e. coli can be tranfected with any DNA plasmid, including the pBLU, which contains the LacZ gene.  Do not use the e. coli for the pGLO activity. Follow the kits instruction to create a plate of pBLU bacteria ahead of time, but instead of adding pGLO, add pBLU to the bacteria. Spread them on a LB plate with ampicillin.  You will also need a plate of bacteria that did not get the pBLU as a control. If you would like to skip this setup, you may contact [email protected] to see if she can send a pretransformed plate of LacZ bacteria, but please keep in mind that this may not be possible..

Label your LB + Amp and LB/AMP/xGal plates with either A or B to disguise their identity in advance of the LacZ biosensor activity.

(5 mins)

Present the idea that cells can be used as biosensors to detect chemicals in their environment. It is recommended to use the robot analogy where by the cell is using sensors to detect chemicals, using its DNA as a program to convert that sensory input into a response.

The first activity is LacZ biosensors, you are welcome to explain the Lac Operon, but we find it also works to explain that the cells have an enzyme that will turn blue if it notices a particular sugar, xGal, in its environment. While this is not entirely true, it demonstrates the basics of biosensors (sense -> response).

Demonstrate proper streaking technique and experimental set up. Student will have to streak their own plates with the bacteria.

Set up Biosensors (20 mins):

Have students label their mystery plates (bottom of the plate, not the lid) by drawing a line to divide the plate in half and labeling one half LacZ+ and one half LacZ-. They should also label the plates with their group name.

Walk around with the two bacterial starter plates and allow students to touch their bacterial loops to a colony and streak their plates.

Tape plates closed and place them in the incubator overnight.

Observe Biosensors (20 mins):

Allow students ample time to investigate their biosensor plates. Use the bacterial biosensor worksheet to help guide their observations and conclusions about the plates.  Optional: allow students to explore the colonies with the dissecting scopes.

Intro to Transformation (20 mins):

Have a few volunteers report out which plate (plate A or B) contained X-gal.

Let the students know they will now be creating their own biosensor from scratch. Present an introduction to transformation. Include a background on the protocol and why each step is important. Present the “map” of the pGLO plasmid and go over what each gene does in the plasmid. 

Bacterial Transformation (60 mins):

Encourage student to follow the instructions given to them on the bacterial transformation worksheet. Once they have completed their bacterial transformations, have them tape all three plates together and place them in the incubator overnight.

Note: in this protocol we use three plates. BioRad’s original protocol uses 4.  We find that our protocol results in less confusion with students after the transformation is completed as it allows for direct comparison of +pGLO and –pGLO bacteria on every type of bacterial plate.

Observe Bacterial Transformation (20 mins):

Allow student to investigate their bacterial transformation. Have them use the bacterial transformation observation worksheet to guide their observations. There should be one UV light included with the BioRad kit, but it is recommended to offer extra UV lights so more than one group at a time can check their plates.

Many students get confused about the conditions that are required for bacteria to glow. Ask students (if they have a successfully glowing colony) why that colony glows? What conditions are met that allow it to glow? Bacteria has pGLO plasmid, arabinose is present, UV light on.

(30 mins)

As a whole class share-out, ask about the conditions on each plate. Which cells survive on LB plates? Which on LB/Amp plates? Why? Which on LB/Amp/Ara plates? Why? Do the cells glow on LB/Amp or LB/Amp/Ara plates when exposed to UV light? Why?

Present a programming analogy for the pGLO experiment. This thought experiment is similar to the mystery program reverse engineering. There are two elements to the program: Ampicillin Resistance and Arabinose Detection. In the pGLO program, ampicillin resistance is always on allowing cells to survive even in the presence of the antibiotic. IF arabinose is detected, THEN bacteria make GFP. A pGLO program in open roberta might look something like the program below:

Structure and Function

Natural Selection and Evolution

Inheritance and Variation of Traits

ETS2 Links Among Engineering, Technology, Science, and Society

HS-LS1-1 From Molecules to Organisms: Structures and Processes

(The plasmid DNA determines the structure of the protein GFP, which allows bacteria to glow. The araC gene acts as a switch to turn the GFP gene on and off in the presence or absence of arabinose, respectively.)

HS-LS3-1 Heredity: Inheritance and Variation of Traits

(The plasmid DNA copying gene “ori” makes sure that the engineered bacterium’s offspring keep the new plasmid DNA.)

HS-LS4-5 Biological Evolution: Unity and Diversity

(When the bacteria with the pGLO plasmid DNA are placed on plates with the antibiotic (new environment), they survive, while bacteria without the pGLO plasmid DNA die off. Therefore the presence of the antibiotic causes the disappearance of a particular trait (a lack of the pGLO plasmid DNA, and therefore non-glowing bacteria.))

HS LS1.A Structure and Function

HS LS3.A Inheritance of Traits

HS LS4.C Adaptation

ETS2.A Interdependence of Science, Engineering, and Technology

(Science and engineering are linked and use similar tools to answer complex questions. In this example, the function of DNA within an engineered bacteria is similar to a program run by a robot. Scientist create and study engineered bacteria just as engineers create and study new programs in robots.)

Practice 3. Planning and Carrying Out Investigations

Practice 4. Analyzing and Interpreting Data

Practice 6. Constructing Explanations and Designing Solutions

Cause and Effect

Structure and Function

Stability and Change