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20.109(F22): Laboratory Fundamentals of Biological Engineering

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       M1: Genomic instability        M2: Drug discovery        M3: Project design       


Though the theme of Module 2 is focused on screening for small molecules that bind the FKBP35 from Plasmodium falciparum (PfFKBP35), today will focus on a few key techniques used in DNA engineering. Because the sequence of proteins is determined by the sequence of the genes that encode them, learning how to manipulate DNA is an important first step. Today you will complete the cloning steps used to incorporate the gene that encodes the PfFKBP35 protein into an expression vector. The expression vector contains the genetic elements needed to express and purify a protein of interest.

Expression vectors contain several features important for cloning, plasmid replication, and protein expression -- all of which are important for purifying high-quality protein. To generate this expression plasmid, two common DNA engineering techniques were used: restriction enzyme digestion and ligation. First, the FKBP35 insert was synthesized such that the gene sequence is flanked by restriction enzymes sites. Next, this fragment and the vector were digested to create compatible ends. Last, the compatible ends of the digested insert and vector were ligated to generate the pET-28a_PfFKBP35 expression plasmid.

Schematic of pET-28a_PfFKBP35 cloning.

Restriction enzyme digest

Schematic of DNA digestion.

Restriction endonucleases, also called restriction enzymes, 'cut' or 'digest' DNA at specific sequences of bases. The restriction enzymes are named according to the prokaryotic organism from which they were isolated. For example, the restriction endonuclease EcoRI (pronounced “echo-are-one”) was originally isolated from E. coli giving it the “Eco” part of the name. “RI” indicates the particular version on the E. coli strain (RY13) and the fact that it was the first restriction enzyme isolated from this strain.

The sequence of DNA that is bound and cleaved by an endonuclease is called the recognition sequence or restriction site. These sequences are usually four or six base pairs long and palindromic, that is, they read the same 5’ to 3’ on the top and bottom strand of DNA. For example, the recognition sequence for EcoRI is 5’ GAATTC 3’ (see figure at right). EcoRI cleaves the phosphate backbone of DNA between the G and A of the recognition sequence, which generates overhangs or 'sticky ends' of double-stranded DNA.

Unlike EcoRI, some other restriction enzymes cut precisely in the middle of the palindromic DNA sequence, thus leaving no overhangs after digestion. The single-stranded overhangs resulting from DNA digestion by enzymes such as EcoRI are called sticky ends, while double-stranded ends resulting from digestion by enzymes such as HaeIII are called blunt ends. HaeIII recognizes 5’ GGCC 3’ and upon recognition cuts in the center of the sequence.


Schematic of DNA ligation.

In a ligation reaction, DNA ends are covalently attached to one another via the ligase enzyme. The efficiency of the reaction is related to type of DNA ends: compatible sticky ends will ligate more efficiently than blunt ends, and non-compatible sticky ends will not be ligated due to the lack of hydrogen bonding between the basepairs. To initiate the ligation reaction, hydrogen bonds are formed between the compatible overhangs of DNA fragments. The ligase enzyme then forms a covalent phosphodiester bond between the 3' hydroxyl end of the 'acceptor' nucleotide and the 5' phosphodiester end of the 'donor' nucleotide.

The first step in this process is the addition of AMP (adenylation) to a lysine residue within the active site of DNA ligase, which releases a pyrophosphate. Next, the AMP is transferred to the 5' phosphate of the donor nucleotide resulting in the formation of a pyrophosphate bond. Lastly, a phosphodiester bond is formed between the 5' phosphate of the donor nucleotide and the 3' hydroxyl of the 3' acceptor nucleotide.


Part 1: Synthesis and restriction enzyme digest of PfFKBP35 insert

Because DNA engineering at the benchtop can take days, if not weeks, you will clone the expression plasmid in silico today. You can use any DNA manipulation software you choose to complete the protocols, but the instructions provided are for SnapGene. Please note that if you use a different program the Instructors may not be able to assist you.

To use SnapGene software off campus you must log into a VPN connection prior to opening the SnapGene. Here is the link to the VPN download and installation instructions. Also you will need to update the SnapGene license number if you have not opened the application since March. The new license information can be found here.

As discussed in the M2 project overview, PfFKBP35 is an essential protein of unknown function in P. falciparum. In an effort to study PfFKBP35 a researcher in the Niles Laboratory, Dr. Khan Osman, cloned the gene that encodes the protein into an expression plasmid. Rather than amplifying the gene from the P. falciparum genome, synthesis technology was used to generate pieces of the gene that were stitched together via 25 bp overhangs. This resulted in a double-stranded DNA segment that was synthesized commercially without the use of live cells. This method is useful for several reasons including: 1. it can be technically difficult to amplify genes from certain organisms, and 2. it can be easier to modify DNA that is synthetically generated. For this project, the PfFKBP35 sequence was modified such that the codon usage was optimized for expression in E. coli cells.

Fa22 M2D1 insert synthesis.png
  1. Open the word document with the PfFKBP35 insert sequence (linked here).
    • Open SnapGene. From the options, select 'New DNA File...'.
    • Copy and paste the sequence from the .docx file above.
    • Enter "PfFKBP35" for the File Name (in the lower, right corner), select 'linear' for the topology (in the lower, left corner), then click 'OK'.
  2. A new window will open with a map of PfFKBP35 showing the unique restriction enzyme sites within the sequence.
  3. In later steps you will generate a map of the PfFKBP35 insert cloned into the pET-28a expression vector. To make the map more visually useful, create a feature that defines the PfFKBP35 insert.
    • Click 'Sequence' from the options at the bottom of the window.
    • Highlight the entire sequence in the window.
    • From the toolbar, select 'Features' → 'Add Feature...'
    • In the new window name, type PfFKBP35 into the 'Feature:' box.
    • Select gene from the dropdown in the 'Type:' box and select the right facing arrowhead (this denotes the directionality of the insert).
    • Then click 'OK'.
  4. The PfFKBP35 sequence was modified such that a 6xHis-tag sequence was added to the N-terminal end of the protein. 6xHis-tags are added for protein purification.
    • Add the 6xHis-tag (5' MGSSHHHHHHSSG 3') (5' ATGGGGTCAAGCCACCATCATCACCATCACTCATCTGGA 3') to the PfFKBP35 insert sequence by setting the cursor to the location in the DNA sequence. Then begin typing the 6xHis-tag sequence. A new window will appear with the typed bases. Confirm the bases are entered correctly, then click 'Insert'.
    • Use the steps above to define the 6xHis-tag sequence as a feature.
  1. In your laboratory notebook, draw a schematic diagram that shows the following:
    • The gene sequence (as a line) with 5' and 3' orientation denoted.
    • The associated protein sequence (again, as a line) with the N' and C' termini denoted.
    • The location of the 6xHis-tag on the gene sequence and protein sequence.
  2. As shown in the schematic of our cloning strategy, NcoI and BamHI recognition sequences were added to the PfFKBP35 sequence to enable cloning into the pET-28a vector. Specifically, NcoI was added to the 5' end and BamHI to the 3' end of the PfFKBP35 sequence.
    • Search the NEB enzyme list to find the NcoI and BamHI recognition sequences.
  3. In your laboratory notebook, complete the following:
    • Record the recognition sequences for NcoI and BamHI. Include the cleavage locations within each sequence.
    • Include the recognition sites for NcoI and BamHI to the schematic diagram created above. Should these recognition sites be included on the gene sequence or the protein sequence?
  4. Add the NcoI and BamHI recognition sites to the PfFKBP35 insert sequence by setting the cursor to the location in the DNA sequence, then begin typing. A new window will appear with the typed bases. Confirm the bases are entered correctly, then click 'Insert'.
  5. Now that you have the PfFKBP35 sequence with the modifications necessary for cloning and purification, you need to digest with NcoI and BamHI to generate 'sticky ends' that will enable you to ligate the PfFKBP35 insert into the pET-28a vector.
    • On the map of the PfFKBP35 insert, select the NcoI recognition site by clicking on the enzyme name. Then hold the shift key and select the BamHI recognition site.
    • This should highlight the area between the enzyme recognition sites.
  6. Click the drop-down arrow next to the 'Copy' icon at the top of the window.
    • Select 'Copy Restriction Fragment.'
  7. Click the drop-down arrow next to the 'New' icon at the top of the window.
    • Select 'New DNA File...'.
    • Paste the restriction fragment from the previous step in the text box, then click 'OK'.
  8. A new window will open with the digested PfFKBP35 insert.
  9. In your laboratory notebook, complete the following:
    • Record the length of the insert. How does the length of the insert compare to the length of the sequence.
    • Is the insert double-stranded or single-stranded?
    • Is the insert a blunt end product or sticky end product?
  10. Save the insert file.

Part 2: Restriction enzyme digest of pET-28a expression vector

For the ligation step, it is important to generate compatible 'sticky ends' on the insert and vector. Above, you digested your PfFKBP35 insert with NcoI and BamHI in a double-digest to prepare the insert for your cloning. Here you will digest the pET-28a vector to create compatible ends that can be ligated.

Fa22 M2D1 vector digest.png
  1. Open the word document with the pET-28a vector sequence (linked here).
    • Copy and paste the vector sequence into a New DNA File window and save this sequence.
    • Be sure to select circular from the topology options.
  2. One very useful aspect of SnapGene is that the software is able to recognize features, or sequences that match known genes and binding sites, in DNA sequences. A window titled "Detect Common Features" should appear.
  3. In your laboratory notebook, include a summary of the details provided about features in the pET-28a vector.
  4. Select 'Add Features'.
  5. A new window will open with a map of the vector showing the unique restriction enzyme sites and annotated features within the sequence.
  6. To generate the sticky ends that will enable you to ligate the PfFKBP35 insert into the vector, view the map of your vector sequence.
    • Select the BamHI recognition site by clicking on the enzyme name, then hold the shift key and select the NcoI recognition site.
    • Select 'Actions' --> 'Restriction and Insertion Cloning' --> 'Delete Restriction Fragment...' from the toolbar.
  7. In your laboratory notebook, complete the following:
    • What is the length of the digested vector product?
    • How many basepairs were removed (compared to the intact cloning vector)?

Part 3: Ligation of PfFKBP35 insert and pET-28a expression vector

Before you prepare a ligation, one very important step is to calculate the amounts of DNA that will be used in the reaction. Ideally, you should use a 3:1 molar ratio of insert to vector (note: it is a molar ratio, not a volumetric ratio!). You will use the steps below to calculate the volume amount (based on the molar ratio!) of the PfFKBP35 insert and pET-28a expression vector you would use to complete this ligation in the laboratory.

Recovery gel for ligation calculations. Lane 1 = pET-28a expression vector, Lane 2 = molecular weight ladder, and Lane 3 = PfFKBP35 insert.

Use the following information to calculate the volume of insert and vector needed to prepare a ligation with a 3:1 molar ratio (insert:vector).

  • Concentration of PfFKBP35 insert solution = 25 ng/uL
  • Concentration of pET-28a expression vector solution = 50 ng/uL
  • Molecular weight of a basepair = 660 g/mol
  • Sizes, in basepairs, of the insert and vector sequences (this was determined in the exercises above!)

Though there are are different strategies that can be used to complete the ligation calculations, it may be easier to break the math into the following steps:

  1. Determine the volume of vector that will be used in the ligation reaction.
    • Typically, it is best to use 50 - 100 ng of vector.
  2. Calculate the moles of vector.
  3. Calculate the moles of insert.
    • Remember, this number should be 3-fold more than the moles of vector to accomplish a 3:1 molar ratio.
  4. Calculate the volume of insert that contains the appropriate moles of insert.
  5. One additional consideration is the volume of the reaction. The total volume of the ligation reaction should not be greater than 15 μL. In this, the total volume of the insert and vector should not be greater than 13.5 μL as additional reagents are required in the reaction.
    • If the insert and vector volume total greater than 13.5 μL, you should (1) scale down both DNA amounts, using less than 50 ng backbone and/or (2) stray from the ideal 3:1 molar ratio.
    • You may ask the teaching faculty for advice during class if you are unsure what choice is best.
  6. In your laboratory notebook, calculate the volume of insert and volume of vector that should be used for a ligation reaction that contains a 3:1 molar ratio of insert:vector. Show all math!
    • Feel free to take a picture of your hand-written work and embed the image in your notebook.
  7. Next you will complete this ligation in silico to generate a map, or visual representation, of the pET-28a_PfFKBP35 plasmid.
    Fa22 M2D1 ligation.png
  8. To ligate the PfFKBP35 insert into the pET-28a expression vector, select 'Actions' --> 'Restriction and Insertion Cloning' --> 'Insert Fragment...'.
    • A new window will open. In the bottom workspace of the window, a cloning schematic will appear showing a vector and insert icon.
    • Click on the 'Vector' label. Then in the workspace at the the right of the window, select the vector file from the 'Vector:' drop-down.
    • Select the restriction enzymes used to digest the expression vector from the drop-down boxes next to the text boxes that contain 'cut'.
  9. Next, click on the 'Insert' label at the bottom of the window and complete the steps as done for the expression vector.
    • For the insert, use the PfFKBP35 undigested file.
    • Make sure you orient your insert so that it is downstream of the T7 promoter. You can change orientation of the insert using the left and right arrows on the bottom right of the window.
  10. Click 'Clone'.
  11. A new window will open with the cloned pET-28a_PfFKBP35 product!
  12. In your laboratory notebook, complete the following:
    • What is the size of the plasmid? Does this make sense given the lengths of the insert and vector?
    • Does your sequence still contain a NcoI recognition sequence? A BamHI recognition sequence?

Part 4: Confirmation digest of pET-28a_PfFKBP35

To confirm the pET-28a_PfFKBP35 construct that we will use for this module, you will perform a 'diagnostic' or 'confirmation' digest. As discussed in prelab, this step is an important control -- you want to be sure that the products you use in your research are correct! This step is used to check products you clone yourself and, perhaps more importantly, those that you may receive from another researcher.

Ideally you will use a single enzyme that cuts once within the vector and once within your insert. Unfortunately, this is rarely an option and you instead need to select an enzyme that cuts once within the vector and a second, compatible enzyme that cuts once within the insert. Enzyme compatibility is determined by the buffer. If two enzymes are active, or able to cleave DNA, in the same buffer, they are compatible. The NEB double digest online tool will prove very helpful in identifying compatible enzyme combinations!

Use the information from prelab, the 20.109 list of enzymes (linked here), and the plasmid map you generated above to choose the enzymes you will use.

  1. To choose restriction enzymes for your confirmation digest, look at the plasmid map for your pET-28a_PfFKBP35 construct.
    • Identify possible sites that will enable to you confirm the pET-28a_PfFKBP35 sequence.
    • Remember the guidelines discussed in prelab!
  2. After you identify the enzymes that you will use for the confirmation digest, complete a virtual digest in using the pET-28a_PfFKBP35 map you generated above.
    • On the map of pET-28a_PfFKBP35, select the first recognition site by clicking on the enzyme name. Then hold the shift key and select the second recognition site.
    • Select 'Tools' --> 'Simulate Agarose Gel' from the toolbar.
  3. In your laboratory notebook, complete the following:
    • Record the expected fragment sizes from the confirmation digest.
    • Are the fragments distinct or ambiguously close together?
  4. Now that you identified which enzyme(s) to use in your confirmation digest, consider which controls should be included to ensure the results are interpretable.
  5. In your laboratory notebook, explain why the following reactions are included as controls for the confirmation digest experiment:
    • Undigested pET-28a_PfFKBP35.
    • Single digests of pET-28a_PfFKBP35 (each enzyme used alone in a digest with pET-28a_PfFKBP35).
  6. Use the table below to calculate the volumes of each reagent that should be included in the confirmation digest reactions.

Keep the following in mind as you consider which enzymes to use:

    • Each enzyme should be present in 10 U quantity per reaction. As an example, the XbaI vial contains 20,000 U/mL, or 20 U/μL.
    • The 20.109 enzyme stocks are always the "S" size and concentration when you search for them on the NEB website.
    • Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.
    • To find the concentration of the enzyme(s) you choose, search the NEB site.

Diagnostic digest
(enzyme #1 AND enzyme #2)
Enzyme #1 ONLY Enzyme #2 ONLY Uncut
(NO enzyme)
pET-28b(+)_PF3D7_20109-F21 5 μL 5 μL 5 μL 5 μL
10X NEB buffer

(buffer name:____________)

2.5 μL 2.5 μL 2.5 μL 2.5 μL
Enzyme #1

(enzyme name:____________)

____ μL ____ μL
Enzyme #2

(enzyme name:____________)

____ μL ____ μL
H2O to a final volume of 25 μL
  1. Unlike the cloning steps you completed above, the diagnostic digest will be performed at the benchtop.
  2. Prepare a mix for each of the above reactions (uncut, cut ONLY with enzyme #1, cut ONLY with enzyme #2, and cut with enzyme #1 AND enzyme #2) that includes (in that order) water, buffer, and enzyme in well-labeled eppendorf tubes.
    • The labels should include the plasmid name, the enzyme(s), and your team color.
  3. Pipet 5 μL of pET-28a_PfFKBP35 into the four well-labeled eppendorf tubes.
  4. Flick the tubes to mix the contents then gather the liquid in the bottom of the tube with a short spin in the microcentrifuge.
  5. Incubate your digests at 37 °C.

The teaching faculty will leave your digests at 37 °C for one hour, then move them to -20 °C.

Reagents list

  • pET-28a_PfFKBP35 (concentration = 25 ng/μL) (a gift from the Niles Laboratory)
  • 10X buffer; the buffer will depend on the enzymes you use for your confirmation digest (from NEB)
  • restriction enzyme(s); the concentration of each enzyme is listed on the product information page (from NEB)
  • 1% agarose in 1X TAE (agarose from VWR)
    • with 10% (v/v) μL SYBR Safe DNA stain (from Invitrogen)
  • 1X TAE gel electrophoresis buffer: 40 mm Tris, 20 mM acetic acid, 1 mM EDTA (from BioRad)
  • 6X gel loading dye, blue (from NEB)
  • 1 kb DNA ladder (from NEB)

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