resources for chapter 4: genetic engineering your e. coli

Lesson

© Science Alberta Foundation 2019. All rights reserved.

From Zero to Genetic enGineerinG Hero - tHe BeGinner’s Guide to ProGramminG Bacteria

by Justin Pahara & Julie Legault (Amino Labs)

Resources for Chapter 4: Genetic Engineering your E. coli cellsThese resources are meant to be used in the order in which they are listed. Times are approximate.

• Chapter opener: How do we change E. coli cells to do something for us?

30 – 60 min with possibility for open-ended whole class discussion (time permitting)

• Introducing Key Knowledge

90 – 120 min (includes time for discussion)

- Introduction - Genetic Engineering PPT

- Introduction - Controls and Variables PPT

- Exit Ticket

• Pre-lab: Virtual Bioengineer 3-7 Worksheet (Key available)

60 – 90 min

• Check Point: Are you ready for engineering E. coli cells? (Key available)

30 min

• Lab: Genetic engineering E. coli – Student handout (Key available)

90 – 120 min

24 – 48 hrs incubation

30 – 60 min viewing of plates and post-lab questions

- Lab: Genetic engineering E. coli – Self Assessment Checklist

• Post Lab: Your Ideas

30 min

2

CHAPTER 4 RESOURCE: OPENER


NAME: CLASS: DATE:

How do we cHanGe e. coli cells to do sometHinG For us?

1. People have been genetically engineering E. coli bacteria for decades. The goal is to produce something useful, for example the hormone insulin for treating diabetes patients. How can we change the bacteria to make it produce a human protein such as insulin? Write and draw your ideas below.

2. In pairs, share and discuss your answer to the previous question. Identify two things about genetic engineering of E. coli that you and your partner are unsure about and write your questions below.

3. Changing living things for humans’ benefit is not a new activity. In fact, this has been going on for millennia. Write some examples in the space below, then share and discuss with your partner.

3

CHAPTER 4 RESOURCE: OPENER


NAME: CLASS: DATE:

4. Changes take place constantly all around us and have been going on since the beginning of time. In this chapter you will learn how to change the genes of E. coli cells. What thoughts does the concept of change bring to your mind? Use the space below to record your thoughts.

5. Read Chapter 4 introduction on page 83. List any terms you were not sure about and look up their meaning.

6. If there is a Questions & Ideas Board in your classroom, post two of your questions and/or ideas from this page – and revisit them at a later time. (Optional)

4

CHAPTER 4 RESOURCE: EXIT TICKET


NAME: CLASS: DATE:

introductory KnowledGe- exit sliP

The drawing below represents a blank E. coli cell. Add labels.

Where are the blueprints (or genes) of this E coli cell located?

How will this E coli cell look after it is genetically engineered? Label your drawing.

5

CHAPTER 4 RESOURCE: EXIT TICKET


NAME: CLASS: DATE:

Below is a Selective LB-agar plate with growing E coli cells. What kind of cells are growing on this plate? Explain your answer.

The DNA added to an engineered E coli has three major components. Name these components and briefly describe their roles. Use a simple sketch to support your answer.

6

CHAPTER 4 RESOURCE: PRE-LAB: VIRTUAL BIOENGINEER STEPS 3-7 WORKSHEET


NAME: CLASS: DATE:

The Virtual Bioengineer Simulation is a free interactive online activity that will help you prepare for the actual hands-on bioengineering lab. It involves the same procedure, materials and equipment as the ones you will use in the real lab, and will take you only 20-30 minutes to complete. Enjoy!

In this pre-lab activity you will focus on the techniques used to genetically engineer E coli cells.

Note: It is assumed you already know how to make LB Agar plates and how to use them to grow E coli.

What you need: Computer with internet connection; Chrome browser

1. Open the Chrome browser

2. Go to this web page https://amino.bio/pages/launchedvbio

3. Click START to launch the simulation.

Choose one of the two options – Red Bio paint or Banana Perfume – and run through the first two steps as you have done in chapter 3. This should provide a good refresher:

• Make LB Agar Plates

• Grow Blank Cells

4. Continue with steps 3 through 7. Fill out the Virtual Bioengineer Step 3-7 Worksheet.

5. Save your Virtual Bioengineer Certificate into your science portfolio

Pre- laB: Virtual BioenGineer stePs 3-7

https://amino.bio/pages/launchedvbio

7



NAME: CLASS: DATE:

Step 3 – MAKE CELLS COMPETENT

1. To engineer the cells, first you must make them competent. What does this mean?

2. List all the materials needed for making cells competent.

3. The transformation buffer consists of water mixed with some special salts. Identify one of its main salts and explain how this salt helps in the transformation process. Use a sketch to support your explanation.

8



NAME: CLASS: DATE:

4. Which station on the DNA Playground is used for making cells competent?

5. The Blank E coli Cells will be obtained from a freshly streaked LB-Agar plate you incubated overnight. Which part of the plate should you collect cells from? Why?

6. Sketch a small group (or colony) of blank E coli cells. How will they differ after a successful transformation?

Before transformation After transformation

7. Where should you place the transformation buffer? What kind of temperature should be used throughout the experiment and why is that important?

9



NAME: CLASS: DATE:

8. How will you mix the cells into the transformation buffer? What should you watch for to ensure you are doing this correctly?

9. Where should you place the cell suspension and how soon after mixing should you perform the transformation?

Step 4 – TRANSFORM CELLS

10



NAME: CLASS: DATE:

10. List the materials needed to transform cells.

11. Before adding the new DNA to the competent cells, make sure the hot station has reached a temperature of ____________. Why is this important?

12. Describe the method you’ll use to collect a small amount of DNA from the DNA tube.

13. Where should you keep the competent cells when mixing them with the DNA?

14. The competent cells must be mixed well with the new DNA. Why is this important?

11



NAME: CLASS: DATE:

15. After mixing, the competent cells & DNA should be kept on the cold station for 10 minutes. What happens during this time? Use a sketch to explain.

16. Next, the mixture is “heat shocked” by transferring it to the hot station for 90 seconds. What happens in this step? Use a sketch to explain.

17. After the 90s heat shock, the cells & DNA mixture must be moved back to the cold station. What is accomplished during this step? Use a sketch to explain.

12



NAME: CLASS: DATE:

Step 5 – RECOVER CELLS

18. List the materials needed for the recovery of cells.

19. The goal of the recovery step is to get the transformed cells growing again. The hot station temperature must be changed to ____________ as this is the optimal temperature for growing E coli.

20. Why should you add Recovery media to the transformed cells?

21. You now have a tube of transformed E coli cells in recovery media. What is the next step to get the cells ready for plating? What happens inside the cells during this step?

13



NAME: CLASS: DATE:

Step 6 – PLATE CELLS

22. List the materials needed.

23. Why should you plate the transformed cells on a Selective LB Agar plate?

24. How will you plate the cells? Describe the procedure.

14



NAME: CLASS: DATE:

25. What controls would you include in this experiment and why? Explain your thinking.

Step 7 – INCUBATE CELLS

26. This is the final step in the genetic engineering of E coli cells. How should you store the LB Agar plate (or plates) during incubation?

27. At what temperature should you incubate the cells?

28. How long until you expect to see cell growth?

29. Why might newly transformed cells grow slower than blank cells?

YOU ARE NOW A VIRTUAL BIOENGINEER!

15



NAME: CLASS: DATE:

The Virtual Bioengineer Simulation is a free interactive online activity that will help you prepare for the actual hands-on bioengineering lab. It involves the same procedure, materials and equipment as the ones you will use in the real lab, and will take you only 20-30 minutes to complete. Enjoy!

In this pre-lab activity you will focus on the techniques used to genetically engineer E coli cells.

Note: It is assumed you already know how to make LB Agar plates and how to use them to grow E coli.

What you need: Computer with internet connection; Chrome browser

1. Open the Chrome browser

2. Go to this web page https://amino.bio/pages/launchedvbio

3. Click START to launch the simulation.

Choose one of the two options – Red Bio paint or Banana Perfume – and run through the first two steps as you have done in chapter 3. This should provide a good refresher:

• Make LB Agar Plates

• Grow Blank Cells

4. Continue with steps 3 through 7. Fill out the Virtual Bioengineer Step 3-7 Worksheet.

5. Save your Virtual Bioengineer Certificate into your science portfolio

Pre- laB: Virtual BioenGineer stePs 3-7 (Key)

https://amino.bio/pages/launchedvbio

16



NAME: CLASS: DATE:

Step 3 – MAKE CELLS COMPETENT

1. To engineer the cells, first you must make them competent. What does this mean?

MAKING CELLS ABLE TO TAKE IN DNA

2. List all the materials needed for making cells competent.

TRANSFORMATION BUFFER, LB AGAR PLATE WITH BLANK E COLI CELLS, STERILE BLUE INOCULATION LOOP, DNA PLAYGROUND

3. The transformation buffer consists of water mixed with some special salts. Identify one of its main salts and explain how this salt helps in the transformation process. Use a sketch to support your explanation.

CALCIUM CHLORIDE

+VE CALCIUM IONS BIND TO NEGATIVELY CHARGED PHOSPHATES ON THE DNA MOLECULES

THIS MAKES THE DNA MORE NEUTRAL AND ABLE TO FOLD INTO A MORE COMPACT FORM BECAUSE THERE ARE FEWER -VE CHARGES REPELLING EACH OTHER

A MORE COMPACT SHAPE HELPS DNA ENTER THE CELLS

17



NAME: CLASS: DATE:

4. Which station on the DNA Playground is used for making cells competent?

THE COLD STATION

5. The Blank E coli Cells will be obtained from a freshly streaked LB-Agar plate you incubated overnight. Which part of the plate should you collect cells from? Why?

FROM ISOLATED WELL SEPARATED COLONIES – THESE CELLS GROW THE FASTEST AMONG ALL CELLS ON THE PLATE BECAUSE THEIR RESOURCES (EG FOOD) ARE STILL PLENTIFUL

FAST GROWING CELLS ARE BEST AT TAKING IN DNA

6. Sketch a small group (or colony) of blank E coli cells. How will they differ after a successful transformation?

Before transformation After transformation

7. Where should you place the transformation buffer? What kind of temperature should be used throughout the experiment and why is that important?

ON COLD STATION – ICE COLD CONDITIONS KEEP SAMPLES STABLE AND PROTECT AGAINST DEGRADATION

18



NAME: CLASS: DATE:

8. How will you mix the cells into the transformation buffer? What should you watch for to ensure you are doing this correctly?

THE LOOP CARRYING THE E COLI CELLS MUST BE IMMERSED INTO THE TUBE WITH TRANSFORMATION BUFFER AND TWISTED VIGOROUSLY UNTIL NO CLUMPS OF CELLS ARE VISIBLE ANYMORE AND THE SUSPENSION LOOKS UNIFORMLY CLOUDY

THE CELLS MUST BE KEPT CALLED AT ALL TIMES

9. Where should you place the cell suspension and how soon after mixing should you perform the transformation?

ON THE COLD STATION – THE SUSPENSION OF COMPETENT CELLS MUST BE TRANSFORMED WITHIN AN HOUR

Step 4 – TRANSFORM CELLS

19



NAME: CLASS: DATE:

10. List the materials needed to transform cells.

TUBE WITH NEW DNA (PLASMID), COLD COMPETENT CELLS (FRESH), STERILE BLUE LOOP, DNA PLAYGROUND

11. Before adding the new DNA to the competent cells, make sure the hot station has reached a temperature of _____ 42 degrees C_______. Why is this important?

THE HOT STATION MUST BE READY FOR THE HEAT SHOCK STEP WHICH HELPS DNA PLASMID ENTER THE COMPETENT CELLS

12. Describe the method you’ll use to collect a small amount of DNA from the DNA tube.

DIP INOCULATING LOOP INTO THE DNA SOLUTION – CHECK THAT THE LOOP CONTAINS A FILM OF LIQUID

13. Where should you keep the competent cells when mixing them with the DNA?

ON THE COLD STATION

14. The competent cells must be mixed well with the new DNA. Why is this important?

MIX THE DNA WITH THE COMPETENT CELLS BY SPINNING THE LOOP INSIDE THE SUSPENSION FOR ABOUT 10 SECONDS. THIS WILL ALLOW THE DNA TO TRANSFER FROM LOOP TO LIQUID AND COME IN CONTACT WITH THE SURFACE OF CELLS

20



NAME: CLASS: DATE:

15. After mixing, the competent cells & DNA should be kept on the cold station for 10 minutes. What happens during this time? Use a sketch to explain.

CALCIUM IONS FLOATING AROUND BIND TO THE DNA AND NEUTRALIZE THE NEGATIVE CHARGES;

THE DNA MOLECULES FOLD INTO COMPACT SHAPES AND ATTACH THEMSELVES TO THE CELLS AND START ENTERING THE CELLS

16. Next, the mixture is “heat shocked” by transferring it to the hot station for 90 seconds. What happens in this step? Use a sketch to explain.

THE CELLS MEMBRANES BECOME MORE FLUID (LIKE PARTIAL MELT) WHICH MAKES IT EASIER FOR DNA MOLECULES TO PASS THROUGH

17. After the 90s heat shock, the cells & DNA mixture must be moved back to the cold station. What is accomplished during this step? Use a sketch to explain.

THE CELLS MEMBRANES BECOME MORE RIGID (THE REVERSE OF WHAT HAPPENS DURING THE HEAT SHOCK) TRAPPING THE DNA INSIDE THE CELLS

21



NAME: CLASS: DATE:

Step 5 – RECOVER CELLS

18. List the materials needed for the recovery of cells.

TUBE WITH RECOVERY MEDIA, TUBE WITH TRANSFORMED CELLS, DNA PLAYGROUND

19. The goal of the recovery step is to get the transformed cells growing again. The hot station temperature must be changed to _____ 37 degrees C_______ as this is the optimal temperature for growing E coli.

20. Why should you add Recovery media to the transformed cells?

THE RECOVERY MEDIA CONTAINS LB MATERIALS (SUGARS, AMINO ACIDS AND MINERALS) THAT E COLI BACTERIA FEED ON; ADDING RECOVERY MEDIA TO THE CELLS HELPS THEM RECOVERING FROM THE STRESS OF THE HEAT SHOCK AND START GROWING AGAIN

21. You now have a tube of transformed E coli cells in recovery media. What is the next step to get the cells ready for plating? What happens inside the cells during this step?

INCUBATE THE TUBE AT 37oC FOR 30-60 MIN; DURING THIS TIME THE CELLS START TO GROW AND DIVIDE AND TO RUN THE NEW DNA PROGRAM I.E. EXPRESS THE GENES ON THE NEW PLASMID: THE ANTIBIOTIC RESISTANCE AND NEW PROTEIN (RED PAINT OR BANANA FLAVOR)

22



NAME: CLASS: DATE:

Step 6 – PLATE CELLS

22. List the materials needed.

SELECTIVE LB AGAR PLATE, TUBE WITH TRANSFORMED CELLS IN RECOVERY MEDIA; YELLOW STERILE INOCULATING LOOP

23. Why should you plate the transformed cells on a Selective LB Agar plate?

TO SELECT FOR NEWLY TRANSFORMED CELLS – ONLY CELLS CARRYING THE NEW DNA WITH THE ANTIBIOTIC RESISTANCE MARKER SHOULD BE ABLE TO GROW

24. How will you plate the cells? Describe the procedure.

BY POURING THE TRANSFORMED CELLS IN THE MIDDLE OF THE SELECTIVE PLATE AND USING THE LOOP TO GENTLY SPREAD THEM OVER THE ENTIRE SURFACE

23



NAME: CLASS: DATE:

25. What controls would you include in this experiment and why? Explain your thinking.

POSITIVE CONTROL – CELLS THAT HAVE BEEN SUCCESSFULLY ENGINEERED AND MUST GROW ONTO THE SAME KIND OF SELECTIVE PLATE (SAME ANTIBIOTIC)

NEGATIVE CONTROL – BLANK CELLS THAT DO NOT CARRY THE ANTIBIOTIC RESISTANCE MARKER AND SHOULD NOT GROW ONTO ON A SELECTIVE PLATE

Step 7 – INCUBATE CELLS

26. This is the final step in the genetic engineering of E coli cells. How should you store the LB Agar plate (or plates) during incubation?

FLIPPED UPSIDE DOWN

27. At what temperature should you incubate the cells?

37 DEGREES C

28. How long until you expect to see cell growth?

24 – 48 HRS

29. Why might newly transformed cells grow slower than blank cells?

BECAUSE THEY NOW HAVE ADDITIONAL DNA PROGRAM TO RUN AND NEW TRAITS TO EXPRESS SUCH AS ANTIBIOTIC RESISTANCE AND THE RED PIGMENT OR BANANA FLAVOR

YOU ARE NOW A VIRTUAL BIOENGINEER!

24

CHAPTER 4 RESOURCE: CHECK POINT: ARE YOU READY FOR ENGINEERING E. COLI CELLS?


NAME: CLASS: DATE:

are you ready For enGineerinG e. coli cells? Use at least two bullet points and labels to describe each step in the protocol. Then, swap with a partner and check each other’s answers.

STEP DESCRIPTIONMake competent cells

Add new DNA to the competent cells

Transform the cells

25



NAME: CLASS: DATE:

Recover the cells

Plate the cells

Set up Controls

26



NAME: CLASS: DATE:

are you ready For enGineerinG e. coli cells? (Key)Use at least two bullet points and labels to describe each step in the protocol. Then, swap with a partner and check each other’s answers.

STEP DESCRIPTIONMake competent cells

• COLLECT BLANK E COLI CELLS FROM 10-20 ISOLATED COLONIES

• ADD THE CELLS TO THE COLD TRANSFORMATION BUFFER

• MIX THOROUGHLY UNTIL NO MORE CELLS CLUMPS ARE VISIBLE – KEEP COLD

Add new DNA to the competent cells

• COLLECT SOME DNA BY DIPPING A LOOP INTO THE DNA TUBE

• ADD THE DNA TO THE CELL SUSPENSION IN TRANSFORMATION BUFFER

• MIX WELL BY TWISTING THE LOOP

• INCUBATE FOR 10 MIN ON COLD

Transform the cells

• HEAT SHOCK: TRANSFER THE TUBE TO 42 DEGREES C FOR 90 SEC

• TRANSFER TUBE BACK ON COLD FOR 2 MIN

27



NAME: CLASS: DATE:

Recover the cells

• ADD RECOVERY MEDIA TO THE TRANSFORMED CELLS

• MIX WELL BY FLIPPING THE TUBE

• INCUBATE AT 37 DEGREES C FOR 30-60 MIN

Plate the cells • POUR THE RECOVERED CELLS ONTO A SELECTIVE PLATE

• USE A STERILE LOOP TO GENTLY SPREAD THE CELLS ONTO THE WHOLE SURFACE

• FLIP AND INCUBATE AT 37 DEGREES C FOR 24-48 HRS

Set up Controls • POSITIVE CONTROL: STREAK POSITIVE CONTROL CELLS ONTO ANOTHER S PLATE (THESE CELLS SHOULD GROW SINCE THEY CONTAIN A DNA PLASMID WITH SAME ANTIBIOTIC MARKER AS THE TRANSFORMED CELLS)

• NEGATIVE CONTROL: STREAK SOME OF THE ORIGINAL BLANK CELLS ONTO A THIRD S PLATE (THESE CELLS SHOULD NOT GROW SINCE THEY DO NOT CONTAIN A PLASMID WITH THE ANTIBIOTIC RESISTANCE)

28

CHAPTER 4 RESOURCE: LAB- GENETIC ENGINEERING E. COLI


NAME: CLASS: DATE:

laB: Genetic enGineerinG e. coli student Handout

Purpose: To insert new DNA into E coli cells so they produce a colored pigment.

Equipment and Materials

• DNA PlaygroundTM - available at Amino Labs https://amino.bio/

• Engineer-it-Kit – available at Amino Labs

• Three Selective (S) LB Agar plates (pre-made; antibiotic must match new DNA and positive control)

• Non-Selective (NS) plate with colonies of Blank E coli (freshly made & incubated overnight)

• Tube with Transformation Buffer – T. Buffer

• Tube with New DNA solution

• Tube with Recovery Media

• Tube with positive control cells

• Sterile inoculation loops – blue and yellow

• Waste container (0.5-1 L)

• Lab gloves (latex or nitrile – disposable); Lab coat or apron; Safety goggles

• Sharpie

• Bleach solution (10% in water)

Procedure:

Making E. coli Cells CompetentSteps Notes and Observations

1. Put on lab gloves, coat and safety goggles

2. Turn ON the cold station on the DNA Playground

3. Obtain a tube with T. Buffer from the refrigerator and place it on the cold station

4. Obtain the NS plate with growing Blank E. coli cells

5. Use a sterile blue inoculation loop to collect Blank E. coli cells from 10-20 well separated colonies

29



NAME: CLASS: DATE:

6. Mix the cells in the COLD T Buffer by twisting the inoculating loop vigorously

7. The competent cells are ready for transformation when the suspension looks evenly cloudy with no visible cell clumps – KEEP COLD

TRANSFORMING THE COMPETENT E. COLI CELLS

8. Turn ON the 42o C hot station on the DNA Playground. Obtain tube with New DNA

9. Dip a sterile blue inoculation loop into the DNA tube and swirl for about 10 seconds. Pull out the loop and make sure that it contains liquid

10. Dip the loop with DNA into the COLD suspension of competent cells. Twist the loop and mix well for 10 seconds

11. Incubate the mixture of competent cells & DNA on the cold station for about 10 minutes

12. Place mixture of competent cells & DNA to the 42o C hot station for 90 seconds

13. Return the mixture to the cold station and let it stand for about 2 minutes

30



NAME: CLASS: DATE:

RECOVERING THE TRANSFORMED E. COLI CELLS

14. Turn ON the 37 degrees C hot station on the DNA Playground. Obtain tube with Recovery Media

15. Pour the Recovery Media into the tube with cells and DNA. Mix well by gently inverting the tube 10 times.

16. Place the tube on the 37 degrees C hot station for 30-60 minutes

17. Turn OFF the cold stationPLATING THE TRANSFORMED CELLS

18. Obtain three Selective (S) LB-Agar plates and label them as follows:

� Transformed

� Positive Control

� Negative Control

Warm up the plates – to room temperature (at least) before using them.

19. Pour the recovered cells onto the plate labeled Transformed. Use a sterile yellow loop to gently spread the cells

20. Obtain the tube with positive control cells. Dip a sterile yellow loop into the tube and streak the positive cells onto the plate labeled Positive Control

31



NAME: CLASS: DATE:

21. Obtain the NS plate with Blank E coli. Use a new sterile yellow loop to collect some Blank cells and streak them onto the plate labeled Negative Control

22. Let the three plates stand for a few minutes or until there is no liquid visible on top of the agar

23. Flip the plates and place them at 37 degrees C in the incubator (agar should be on top)

24. Clean up and use bleach solution to inactivate all materials that came into contact with the bacteria

25. Incubate at 37 degrees C for at least 24-48 hours. Cell growth should become visible after 12 – 16 hours. The new color pigment should start showing after about 24 hours and become stronger after 48 hours and beyond.

26. Store the plates with growing bacteria up-side-down in the refrigerator.

Post Lab Questions:

Answer individually and feel free to add drawings and diagrams as needed. Discuss your answers with a partner, and then revise them as you see fit.

1. What was the purpose of this lab?

2. What kind of E coli cells did you use and why did you have to make them “competent”?

32



NAME: CLASS: DATE:

3. Cells from well isolated colonies work best for making competent cells. Why?

4. To become competent, cells must be mixed with Transformation buffer. What is the composition of the T buffer and how do its ingredients help?

5. Were you successful in making competent cells? What should you watch for when making cells competent? Explain your answer.

6. Cells are said to be competent when they are ready for transformation. What changes occur during transformation? Use a sketch to help you explain.

7. What are the steps in the transformation protocol? Fill out the following flowchart.

Competent cells +

1. _____________________________________

33



NAME: CLASS: DATE:

2. _____________________________________

3. _____________________________________

Transformed E. coli Cells

8. The transformation process is stressful for cells. What did you do to help cells recover?

9. What kind of LB-Agar plate did you use to grow the transformed cells? Why?

10. Did your experiment work? ______

Explain your answer and draw your results below

Transformed Positive Control Negative Control

34



NAME: CLASS: DATE:

11. What will you do differently next time?

Complete the Self-assessment Checklist and score yourself.

35



NAME: CLASS: DATE:

laB: Genetic enGineerinG e. coli student Handout (Key)Purpose: To insert new DNA into E coli cells so they produce a colored pigment.

Equipment and Materials

• DNA PlaygroundTM - available at Amino Labs https://amino.bio/

• Engineer-it-Kit – available at Amino Labs

• Three Selective (S) LB Agar plates (pre-made; antibiotic must match new DNA and positive control)

• Non-Selective (NS) plate with colonies of Blank E coli (freshly made & incubated overnight)

• Tube with Transformation Buffer – T. Buffer

• Tube with New DNA solution

• Tube with Recovery Media

• Tube with positive control cells

• Sterile inoculation loops – blue and yellow

• Waste container (0.5-1 L)

• Lab gloves (latex or nitrile – disposable); Lab coat or apron; Safety goggles

• Sharpie

• Bleach solution (10% in water)

Procedure:

Making E. coli Cells CompetentSteps Notes and Observations

1. Put on lab gloves, coat and safety goggles

2. Turn ON the cold station on the DNA Playground

3. Obtain a tube with T. Buffer from the refrigerator and place it on the cold station

4. Obtain the NS plate with growing Blank E. coli cells

5. Use a sterile blue inoculation loop to collect Blank E. coli cells from 10-20 well separated colonies

36



NAME: CLASS: DATE:

6. Mix the cells in the COLD T Buffer by twisting the inoculating loop vigorously

7. The competent cells are ready for transformation when the suspension looks evenly cloudy with no visible cell clumps – KEEP COLD

TRANSFORMING THE COMPETENT E. COLI CELLS

8. Turn ON the 42o C hot station on the DNA Playground. Obtain tube with New DNA

9. Dip a sterile blue inoculation loop into the DNA tube and swirl for about 10 seconds. Pull out the loop and make sure that it contains liquid

10. Dip the loop with DNA into the COLD suspension of competent cells. Twist the loop and mix well for 10 seconds

11. Incubate the mixture of competent cells & DNA on the cold station for about 10 minutes

12. Place mixture of competent cells & DNA to the 42o C hot station for 90 seconds

13. Return the mixture to the cold station and let it stand for about 2 minutes

37



NAME: CLASS: DATE:

RECOVERING THE TRANSFORMED E. COLI CELLS

14. Turn ON the 37 degrees C hot station on the DNA Playground. Obtain tube with Recovery Media

15. Pour the Recovery Media into the tube with cells and DNA. Mix well by gently inverting the tube 10 times.

16. Place the tube on the 37 degrees C hot station for 30-60 minutes

17. Turn OFF the cold stationPLATING THE TRANSFORMED CELLS

18. Obtain three Selective (S) LB-Agar plates and label them as follows:

� Transformed

� Positive Control

� Negative Control

Warm up the plates – to room temperature (at least) before using them.

19. Pour the recovered cells onto the plate labeled Transformed. Use a sterile yellow loop to gently spread the cells

20. Obtain the tube with positive control cells. Dip a sterile yellow loop into the tube and streak the positive cells onto the plate labeled Positive Control

38



NAME: CLASS: DATE:

21. Obtain the NS plate with Blank E coli. Use a new sterile yellow loop to collect some Blank cells and streak them onto the plate labeled Negative Control

22. Let the three plates stand for a few minutes or until there is no liquid visible on top of the agar

23. Flip the plates and place them at 37 degrees C in the incubator (agar should be on top)

24. Clean up and use bleach solution to inactivate all materials that came into contact with the bacteria

25. Incubate at 37 degrees C for at least 24-48 hours. Cell growth should become visible after 12 – 16 hours. The new color pigment should start showing after about 24 hours and become stronger after 48 hours and beyond.

26. Store the plates with growing bacteria up-side-down in the refrigerator.

Post Lab Questions:

Answer individually and feel free to add drawings and diagrams as needed. Discuss your answers with a partner, and then revise them as you see fit.

1. What was the purpose of this lab?

TO GENETICALLY ENGINEER E COLI BACTERIA SO THEY BECOME COLORED

TO INTRODUCE A NEW DNA (PROGRAM/PLASMID) INTO E COLI CELLS TO MAKE THEM PRODUCE A NEW COLOR PIGMENT

2. What kind of E coli cells did you use and why did you have to make them “competent”?

THE CELLS USED WERE BLANK WHICH MEANS THEY CONTAINED ONLY THE BACTERIAL GENOME (OR CHROMOSOME) AND NO PLASMID

THE BLANK CELLS WERE MADE “COMPETENT” WHICH MEANS THEY WERE “PRIMED” TO TAKE IN NEW DNA

39



NAME: CLASS: DATE:

3. Cells from well isolated colonies work best for making competent cells. Why?

CELLS FROM ISOLATED COLONIES GROW VERY WELL AND VERY FAST. THIS MAKES THEM VERY GOOD AT TAKING IN NEW DNA

4. To become competent, cells must be mixed with Transformation buffer. What is the composition of the T buffer and how do its ingredients help?

THE TRANSFORMATION BUFFER IS A SOLUTION OF VARIOUS SALTS IN WATER; ONE OF THE SALTS IS CALCIUM CHLORIDE; THE +VE CALCIUM IONS BIND TO -VELY CHARGED PHOSPHATES ON THE DNA; THIS RESULTS IN THE DNA BEING MORE NEUTRAL OVERALL AND ABLE TO FOLD INTO A MORE COMPACT SHAPE WHICH HELPS IT ENTER THE CELLS

5. Were you successful in making competent cells? What should you watch for when making cells competent? Explain your answer.

ANSWERS WILL VARY:

SUCCESS - PROCEDURE FOR MAKING CELLS COMPETENT WORKED BECAUSE ALL THREE S PLATES LOOKED AS EXPECTED: (1) THE TRANSFORMED CELLS RESULTED IN COLONIES OF THE EXPECTED COLOR; (2) THE POSITIVE CONTROL CELLS GREW AND PRODUCED EXPECTED PIGMENT; (3) THE BLANK CELLS DID NOT GROW ON THE PLATE WITH ANTIBIOTIC

NO SUCCESS - THE PROCEDURE FOR MAKING CELLS COMPETENT DID NOT WORK BECAUSE THE PLATE WITH TRANSFORMED CELLS DID NOT HAVE ANY COLONIES WHILE THE CONTROLS LOOKED AS EXPECTED

NOT SURE – ALL THREE PLATES HAD BACTERIA GROWING ON THEM WHICH MEANS THE ANTIBIOTIC SELECTION DID NOT WORK (OTHERWISE BLANK CELLS COULDN’T HAVE GROWN)

6. Cells are said to be competent when they are ready for transformation. What changes occur during transformation? Use a sketch to help you explain.

CELLS TAKE IN NEW DNA PLASMID

40



NAME: CLASS: DATE:

7. What are the steps in the transformation protocol? Fill out the following flowchart.

Competent cells + DNA

1. _________COLD- 10 MIN HEAT SHOCK____

2. ________42 DEGREES C- 90 SEC _______

3. ________COLD- 2 MIN_________________

Transformed E. coli Cells

8. The transformation process is stressful for cells. What did you do to help cells recover?

ADDED RECOVERY MEDIA (CONTAINING LB) TO THE TUBE WITH TRANSFORMED CELLS AND INCUBATED THEM AT 37o C FOR AT LEAST HALF HOUR; DURING THIS TIME THE CELLS START GROWING AND DIVIDING – THE GENES ON THE NEW DNA PLASMID START BEING EXPRESSED (THE NEW PIGMENT AND THE ANTIBIOTIC RESISTANCE)

9. What kind of LB-Agar plate did you use to grow the transformed cells? Why?

A SELECTIVE PLATE – A PLATE CONTAINING THE ANTIBIOTIC THAT THE NEW PLASMID CARRIES RESISTANCE AGAINST

41



NAME: CLASS: DATE:

10. Did your experiment work? _ YES/NO/NOT SURE___ SEE QUESTION #5_____

Explain your answer and draw your results below

Transformed Positive Control Negative Control

11. What will you do differently next time?

ANSWERS WILL VARY E.G. TROUBLESHOOT TRANSFORMATION PROTOCOL – VARIOUS POINTS; MAKE FRESH SELECTIVE PLATES

Complete the Self-assessment and score yourself.

42

CHAPTER 4 RESOURCE: POST-LAB: YOUR IDEAS


NAME: CLASS: DATE:

Post-laB: new Plasmid- wHat are your ideas? The same procedure you used for engineering E coli cells to produce color pigments can be applied to make bacteria do all sorts of other things. The only difference will be the new plasmid DNA used to transform the blank cells. As you are learning genetic engineering, ask yourself:

• What new plasmid would you use, and why?

Use the table below to record your ideas – see example in the top row.

What new plasmid would you use? Why? Plasmid DNA Sketch add labelsPlasmid for making red paint To make large amounts of

biodegradable non-toxic paint

Gene for resistance to specific antibiotic

Gene for producing red paint/protein

Origin of replication

CHAPTER 4 RESOURCE: INTRODUCTION GENETIC ENGINEERING

1© Science Alberta Foundation 2019. All rights reserved.

Teacher Notes:

1. Allow students to answer and collect their answers on the board.

2. Discuss and guide to simplest correct answer: GENETICALLY ENGINEERING CELLS MEANS CHANGING THEIR GENES.

3. When students arrive to correct answer switch to next slide

What does it mean “to genetically engineer cells”?Genetic engineering your E. coli cells



GENETICALLY ENGINEERING CELLS means

CHANGING THEIR GENES

What are genes?

Teacher Notes:


2. Discuss and guide to simplest correct answer: GENES ARE BUNDLES OF INFORMATION USED BY ALL LIVING CELLS. THE INFORMATION IS CODED IN THE DNA WHICH WORKS AS A BLUEPRINT.




GENES ARE BUNDLES OF INFORMATION USED BY ALL LIVING CELLS.

THE INFORMATION IS STORED IN DNAWHICH WORKS AS A BLUEPRINT.

Which parts of E. coli have genes?

Teacher Notes:


2. Discuss and guide to simplest correct answer: E COLI GENES ARE FOUND ON (1) BACTERIAL CHROMOSOME (OR GENOME) AND (2) PLASMID




PARTS OF E COLI CELLS THAT HAVE GENES

• Genome (or chromosome)• Plasmid

Plasmid

Teacher Notes:

1. Plasmid-

� Separate from the bacterial chromosome

� Replicates independently

� The part that gets changed in genetically engineered E coli

� Transferable and mobile – similar to a memory stick



Plasmid

ORIGIN OF REPLICATION

NEW TRAIT

ANTIBIOTIC RESISTANCE

Teacher Notes:

1. A plasmid has three main parts-

� ORI – Origin of replication – Ensures plasmid replicates independently and effectively

� TRAIT GENE – This is the DNA code that enables the engineered E coli to make something new

� SELECTION GENE – Allows the engineered E coli to grow in the presence of a selection agent e.g. an antibiotic



What’s this?

Student assignmentImagine you are telling your parents what you’ve been learning about genetic engineering. Your little sister who’s only seven hears you talk and she gets curious. She asks you the following questions.

• What is E coli?• What does it mean to engineer E coli and why do you do that? • What is a plasmid?• Why do you need antibiotics?• Is this safe?

How would you answer her questions and help her understand?

Teacher Notes:

1. Read information on page 93 (Plasmid Going Deeper 4-4) – if needed

2. Students can choose how to explain.

3. Arrange for Students explanations/answers to be provided directly to grade one students – one-on-one – and then quiz the grade ones

CHAPTER 4 RESOURCE: INTRODUCTION CONTROLS AND VARIABLES


Teacher Notes:

1. Allow students to share their ideas and write them up on the board.

2. Collect and Discuss everyday Observations and related Questions: E.g. why is the pot plant wilting, why do some foods mold faster than others, why do I feel more energized on a bright day etc.

3. Guide students through the reasoning process. Help them discover the role of VARIABLES and why good tests/experiments include CONTROLS

CONTROLSWhy do we need them?



Teacher Notes: 1. VARIABLES-

� Amount of water and salt relative to the size of strawberry

� Method used to ‘massage’ the ziplock bag

� Amount of shampoo

� Shampoo type

� Type of filter paper

2. POSSIBLE EXPLANATIONS FOR SEEING NO DNA-

� Strawberry did not mash up properly in the salt water (cells still clumped)

� Cells did not break open properly with the shampoo (DNA still inside cells and attached to cell debris)

� Not a lot of DNA in the filtrate (DNA molecules were trapped in the filter paper)

DNA extraction Lab

• What are some of the variables in this lab?

• Assume no DNA precipitate was visible

What are some possible explanations? How would you test them?



Teacher Notes:

1. VARIABLES-

� Amount of water

� Temperature of water

� Amount of LB Agar

� Extent of dissolving/mixing

� Possible explanations for why the plates do not solidify

Making LB Agar Plates


• Assume the LB Agar plates do not solidify

• What are some possible explanations? How would you test them?



Growing E coli cells


• Assume no cells are growing• What are some possible

explanations? How would you test them?



Clean up and inactivation

• What are some variables in this?

• Are you sure you killed all the bacteria? How would you test this?



VARIABLES – Factors which change (or vary) and thereby affect the outcome of an experiment

CONTROLS – Methods that help us understand and control the effect of variables; they help us make sense of the outcome of an experiment



NAME: CLASS: DATE:

Self ASSeSSmentWhat did I learn?

Complete the self-assessment below. Be sure to add evidence statements to account for each score.

Not confident yet Somewhat confident Confident Self Score

1.I know how to prepare competent E. coli cells

/ 10

2.I know how to transform competent cells

/ 10

3.I know how to recover and grow the transformed cells

/ 10

4.I know why transformed cells must be plated onto Selective LB-agar

/ 10

5.I know how to set up proper controls

/ 10

6.I know how to use sterile technique

/ 10

7.I know how to incubate and store LB agar plates

/ 10

Things I will do to improve my skills and understanding:

resources for chapter 4: genetic engineering your e. coli

Documents