Before you begin:

Learn how to get started and sign up with Benchling here. Start this worksheet using your free academic account in order to get the most out of this worksheet. DNA sequences can be copied directly into your Benchling account. For notebook entries, you can manually copy & paste the content into a new blank entry.

Content and materials for this module were co-developed with Dr. Philip Leftwich, Biology Lecturer at the University of East Anglia (Norwich, UK)


Overview:

We’ll discuss how Benchling can be used to carry out a bacterial transformation in the lab and to confirm if it was successful. Bacterial transformation is a method for introducing exogenous genetic material into a bacterium and typically involves the uptake of a plasmid of interest into its genome. Bacterial cells that successfully incorporate the plasmid are subsequently subjected to antibiotic selection or other analytical methods like Sanger sequencing.

Prior knowledge:

A foundation for different methods of gene transfer in bacteria and familiarity with cell culture techniques and molecular cloning is helpful. You will also need to be aware of how DNA sequences are analyzed through common sequencing methods.

Before you get started on this module, you should also know how to perform molecular cloning. Check out this previous lesson on Molecular Cloning Methods where we discuss different ways to do this on Benchling. This module will assume you’ve already assembled a recombinant plasmid prior to your bacterial transformation experiment.

Learning outcomes:

  • Record experimental information relevant for transformation (selection markers, cell competency, and more)

  • Link bacterial transformation workflows between Benchling Notebook and Molecular Biology

  • Verify cloning constructs through sequencing alignments

Worked Example:

Prepare appropriate reagents for the transformation

Performing bacterial transformations will require you to know a number of related experimental factors like manufacturer source, competent cell type, or antibiotic resistance. This information is important to document to prevent mistakes and carry out experiments.

  • Competent cell source - Indicates if your cells were from a commercial kit or made elsewhere

  • Competent cell type - Most cells are either chemically-competent or electrocompetent

  • Antibiotic resistance - Depending on your sequence, there will be resistance marker(s)

You can easily record this information within the Benchling Notebook. You can use drop-down lists to fill out or type in this information. For transformation reactions with different plasmids, you can even link each one to the DNA sequence using @ mentions. Don’t forget to write down your transformation protocol or attach a reference if you’re using one.

Question(s):

Your transformation protocol instructs you to prepare two LB agar plates with the appropriate antibiotic for each transformation reaction that you’re performing. Use the image above of your experimental notes to answer the following questions:

1. If each plate requires 20 mL of liquid, what is the volume of LB agar solution that you need to prepare?

2. If you have an antibiotic stock solution at a concentration of 50 mg /mL but want the LB agar working solution to be at a final concentration of 100 ug / mL for the volume of liquid you calculated in question 1, how much of the stock solution of the antibiotic do you add?

3. In practice, it’s always important to make more media than you think you need. You could always spill some or make small errors in measurement. Automate your calculations using this Benchling calculator. Just copy the table and its formulas and paste it into a new blank entry. Modify your answers for Q1 and Q2 so the volume of your solutions accounts for one extra LB agar plate.

Note:

The formula for calculating a dilution is C1*V1 = C2*V2 where...

  • C1 is the concentration of the starting solution.

  • V1 is the volume of the starting solution.

  • C2 is the concentration of the final solution.

  • V2 is the volume of the final solution.

Here:

C1 = 50 mg/ml

V1 = ?

C2 = 100 ug/ml

V2 = the volume calculated from question 1.

Hint: Don’t forget to convert between mg and ug !

Try to answer this question on your own and check the "Solution" at the bottom.

Practice:

Now let’s continue practicing how you would prep for a bacterial transformation experiment if you had knowledge of the plasmid sequence you would use.

  • Take a look at this sequence where you’ve cloned the JfyP gene into pET-31b vector and create a copy of this sequence and store it in a Project you have access to.

  • With the sequence open, click into the “Annotations” sub-panel on the right of the screen and look through existing annotations to see what antibiotic resistance marker this plasmid contains.

  • Navigate to the global “Create” button on the left side panel, open a new notebook entry, and label it “Bacterial Transformation” with your initials

  • Now insert a data table with the same headers from previous transformation examples (Plasmid Sequence, Cell Source, Competent Cell Type, and Antibiotic Resistance)

  • Fill in the fields for the data table based on your investigation of the plasmid sequence and assume you’ll be using chemically-competent TOP10 bacteria cells.

Stretch Yourself:

Verify the identity of transformants through DNA sequencing

Antibiotic selections will indicate if appropriate resistance was conferred to the bacteria by transforming your plasmid of interest. However, they don’t provide verification that the identity of your plasmid sequence is accurate. One way to do this is by DNA sequencing.

Experimentally, you need to pick single colonies from your LB agar plate, grow each in liquid culture, and extract DNA from these cells containing your transformed plasmid. Afterward, you can send the DNA for sequencing and will receive results that can be aligned to your expected construct in silico.

Try out the exercise below to verify that both your cloning and transformation experiments were successful.

  • Download these sequencing trace files (Format is .ab1) and store them somewhere easy to locate on your computer.

  • Open your copy of the pET-31b_JfyP sequence and navigate to the “Alignments” sub-panel on the right of the screen and click on the blue “Create New Alignment” button.

  • Select “CHOOSE FILE(S)” and upload the sequencing files you downloaded earlier and afterward, click on the teal “Create Alignment” button.

  • Analyze your newly created alignment and remember which colonies contain the correct sequence. Use the orange bar to view parts of your alignment and/or resize how many bases you’re viewing. (Alternatively, you can also hold down the “Shift” key and scroll with your mouse wheel to scroll horizontally.)

  • Go to “Export” -> “Copy Link” and navigate back into your Notebook entry from earlier. Create a new column header for “Sequencing Results” and paste in the link you copied earlier.

Solution(s):

1. There are 3 transformation reactions so you will need 6 LB agar plates in total. Each plate requires 20mL of liquid therefore you will need to prepare at least 6 * 20 = 120mL of LB agar solution.

Note: In practice, you may want to make a bit more than 120 mL (~140 mL) just in case there is any spillage or mistakes in measurement. Having at least 10% extra is a good rule of thumb!

2. Using the formula, C1 V1 = C2 V2, set up the equation so that we are solving for V1.

V1 = (C2*V2) / C1

C2 = 100 ug/ml or 0.1 mg/mL

V2 = 120 mL

C1 = 50mg/ml

0.1*120 = 12

12/50 = 0.24 mL of antibiotic stock solution

You would add this to (120-0.24) 119.76 mL of LB agar to make a total volume of 120 mL.

3. We actually want to make sure we have some spare working solution in case of spills or pipetting errors. This is easily modified by swapping the value of V2 from 120 to 140 mL.

Now:

(0.1*140) / 50 = 0.3 mL of antibiotic stock solution

You would add this to (140-0.3) 139.7 mL of LB agar to make a total volume of 140 mL

Congrats! You've finished the learning module: Bacterial Transformation.

What’s next?

Interested in how to interpret sequencing data or results? ? Learn how to do better analysis with our next module on DNA Sequencing.

Or learn more about Benchling for Education.

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