Difference between revisions of "20.109(F20):M2D1"

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(Part 3: Ligation of PF3D7_1351100 insert and pET-28b(+) expression vector)
(Part 3: Ligation of PF3D7_1351100 insert and pET-28b(+) expression vector)
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#Be sure to record all of your work for the ligation calculations in your notebook.
 
#Be sure to record all of your work for the ligation calculations in your notebook.
 
#*Feel free to take a picture of your hand-written work and embed the image in your notebook.
 
#*Feel free to take a picture of your hand-written work and embed the image in your notebook.
#Next you will complete this ligation ''in silico'' to generate a plasmid map of your pET_MBP_SNAP_TDP43-RRM12 plasmid.[[Image:Sp20 M1D1 insert, vector.png|thumb|right|400px|]]
+
#Next you will complete this ligation ''in silico'' to generate a plasmid map of your pET_MBP_SNAP_TDP43-RRM12 plasmid.[[Image:Fa20 M2D1 expression plasmid ligation.png|thumb|right|400px|]]
 
#To ligate you TDP43-RRM12 insert into the pET_MBP_SNAP expression vector, select 'Actions' --> 'Restriction and Insertion Cloning' --> 'Insert Fragment...'.
 
#To ligate you TDP43-RRM12 insert into the pET_MBP_SNAP 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.
 
#*A new window will open.  In the bottom workspace of the window, a cloning schematic will appear showing a vector and insert icon.

Revision as of 20:42, 16 July 2020

20.109(F20): Laboratory Fundamentals of Biological Engineering

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Fall 2020 schedule        FYI        Assignments        Homework        Communication |        Accessibility

       M1: Genomic instability        M2: Drug discovery        M3: Metabolic engineering       


Introduction

Though the theme of Module 1 is focused on screening for small molecules that bind the PF3D7_1351100 protein, 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 PF3D7_1351100 protein into an expression vector. The expression vector contains the genetic elements needed to express and purify of a protein of interest.

Expression vectors contain several features important for 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 PF3D7 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-28b(+)_PF3D7_1351100 expression plasmid.

Schematic of pET-28b(+)_PF3D7_1351100 cloning.

Restriction enzyme digest

Schematic of restriction enzyme digest with EcoRI.

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.

Ligation

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.

Protocols

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.

Part 1: Synthesis and restriction enzyme digest of PF3D7_1351100 insert

The PF3D7_1351100 protein is ...

To clone the gene that encodes the PF3D7_1351100 protein, a gBlock was synthesized. ...and codon usage of sequence was optimized for expression in E. coli...

Fa20 M2D1 insert synthesis and digest .png
  1. Open the word document with the PF3D7_1351100 insert sequence (linked here).
    • Open SnapGene. From the options, select 'New DNA File...'.
    • Copy and paste the sequence from the .docx file above.
    • Enter "PF3D7_1351100" 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 PF3D7_1351100 showing the unique restriction enzyme sites within the sequence.
  3. In later steps you will generate a map of the PF3D7_1351100 insert cloned into the pET-28b(+) expression vector. To make the map more visually useful, create a feature that defines the PF3D7_1351100 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 PF3D7_1351100 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 PF3D7_1351100 gBlock 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.
  5. 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.
  6. Add the 6xHis-tag (5' MGSSHHHHHHSSG 3') to the PF3D7_1351100 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.
  7. As shown in the schematic of our cloning strategy, NcoI and BamHI recognition sequences were added to the PF3D7_1351100 gBlock to enable cloning into the pET-28b(+) vector. Specifically, NcoI was added to the 5' end and BamHI to the 3' end of the PF3D7_1351100 sequence.
    • Search the NEB list to find the NcoI and BamHI recognition sequences.
  8. 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?
  9. Add the NcoI and BamHI recognition sites to the PF3D7_1351100 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'.
  10. Now that you have the PF3D7_1351100 gBlock, you need to digest with NcoI and BamHI to generate 'sticky ends' that will enable you to ligate the PF3D7_1351100 insert into the pET-28b(+) vector.
    • On the map of the PF3D7_1351100 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.
  11. Click the drop-down arrow next to the 'Copy' icon at the top of the window.
    • Select 'Copy Restriction Fragment.'
  12. 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'.
  13. A new window will open with the digested PF3D7_1351100 insert.
  14. 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 gBlock.
    • Is the insert double-stranded or single-stranded?
    • Is the insert a blunt end product or sticky end product?
  15. Save the insert file.

Part 2: Restriction enzyme digest of pET-28b(+) expression vector

For the ligation step, it is important to generate compatible 'sticky ends' on the insert and vector. Above, you digested your PF3D7_1351100 insert with NcoI and BamHI in a double-digest to prepare the insert for your cloning. Here you will digest the pET-28b(+) vector to create compatible ends that can be ligated.

Fa20 M2D1 vector digest.png
  1. Open the word document with the pET-28b(+) 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-28b(+) 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 PD3D7_1351100 insert into the vector, view the map of your vector sequence.
    • Select the NcoI recognition site by clicking on the enzyme name, then hold the shift key and select the BamHI 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 PF3D7_1351100 insert and pET-28b(+) expression vector

When you complete a ligation at the bench, one very important step is to calculate the amounts of DNA you will use in the reaction. Ideally, you would use a 3:1 molar ratio of insert to vector (also referred to as the backbone), and would need to calculate how much volume of each solution to use. You can use the steps below to calculate the amount of TDP43-RRM12 insert and pET_MBP_SNAP expression vector you would use to complete this ligation in the laboratory.

Recovery gel for ligation calculations. Lane 1 = TDP43-RRM12 insert, Lane 2 = molecular weight ladder, and Lane 3 = pET_MBP_SNAP expression vector.
  1. The concentrations for the insert and vector were measured using a nanodrop.
    • TDP43-RRM12 insert = 25 ng/uL
    • pET_MBP_SNAP expression vector = 50 ng/uL
  2. Convert the mass concentration to a molar concentration, using the fact that a typical DNA base is 660 g/mol. This conversion will mostly cancel out between the insert and the backbone, except for the difference in number of bases. Feel free to either omit steps that will cancel if you are comfortable doing so, or to keep them if you follow the math better that way.
    • Hint: you need to know the number of basepairs in the backbone and insert. Use your text sequences and/or snap gene files.
  3. Ideally, you will use 50-100 ng of backbone in the this ligation.
    • Referring to the mass concentration, what volume of DNA will this amount require?
  4. Ideally, you will use a 3:1 molar ratio of insert to backbone.
    • Referring to the molar concentrations, how much insert do you need per μL of backbone?
  5. A 15 μL scale ligation should not include more than 13.5 μL of DNA because you must leave enough volume to add buffer and the ligase enzyme.
    • If your backbone and insert volumes total to greater than this amount, you must (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. Be sure to record all of your work for the ligation calculations in your notebook.
    • 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 plasmid map of your pET_MBP_SNAP_TDP43-RRM12 plasmid.
    Fa20 M2D1 expression plasmid ligation.png
  8. To ligate you TDP43-RRM12 insert into the pET_MBP_SNAP 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 PCR amplicon file.
  10. Click 'Clone'.
  11. A new window will open with the cloned final pET_MBP_SNAP_TDP43-RRM12 product!
    • 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 BamHI recognition sequence? An EcoRI recognition sequence? Explain.

Part 4: Confirmation digest of pET-28b(+)_PF3D7_1351100

To confirm the pET_MBP_SNAP_TDP43-RRM12 construct that we will use for this module, you will perform a 'diagnostic' or 'confirmation' digest. Recall from lecture that this step is important as a control -- you want to be sure that the products you use in your research are correct. This is an important step 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 able to function (cleave DNA) in the same buffer, they are compatible. The NEB double digest online tool will prove very helpful!

Use information from the lecture, the 20.109 list of enzymes 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_MBP_SNAP_TDP43-RRM12 ligation product.
    • Identify possible sites that will enable to you confirm the ppET_MBP_SNAP_TDP43-RRM12 sequence.
    • Remember the guidelines discussed in lecture!
  2. After you decide on the enzymes you will use for your confirmation digest, generate a virtual digest in SnapGene.
    • On the map of pET_MBP_SNAP_TDP43-RRM12, 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.
    • Record the expected fragment sizes from the digest in your laboratory notebook.
    • Are the fragments distinct or ambiguously close together?

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.
  • Because the lower limit of your P20 pipet is 2.0 μL, you may need to use the P2 at the front bench for smaller volumes.
  • 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.

The following table may be helpful as you plan your work:

Diagnostic digest
(enzyme #1 AND enzyme #2)
Enzyme #1 ONLY Enzyme #2 ONLY Uncut
(NO enzyme)
pET_MBP_SNAP_TDP43-RRM12 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_MBP_SNAP_TDP43-RRM12 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-28b(+)_PF3D7_1351100 (concentration: 25 ng / μL)
  • 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)

Navigation links

Next day: Perform protein purification protocol

Previous day: Complete data analysis using statistical methods