Difference between revisions of "20.109(S24):M2D2"

Introduction

In the previous laboratory session you chose the strategy you wanted to use to capture cadmium with a cell surface peptide, and generated primers to insert the DNA sequence for your peptide into the YSD vector. Today you will perform the mutagenesis procedure necessary to insert the peptide DNA sequence into our expression vector. To accomplish this insertion, we will use the Q5 Site-Directed Mutagenesis (SDM) kit from NEB. While there are multiple approaches to DNA mutagenesis, this kit offers the advantage of reliable generation of mutants while still being relatively cost effective. This SDM kit utilizes PCR with the specialized primers you designed to introduce the insertion and amplify the resulting plasmid.

Before we continue, we should review the process used to generate actual primers that are used to amplify DNA as part of this process. Current oligonucleotide, or primer, synthesis uses phosphoramidite monomers, which are simply nucleotides with protection groups added. The protection groups prevent side reactions and promote the formation of the correct DNA product. The DNA product synthesis starts with the 3'-most nucleotide and cycles through four steps: deprotection, coupling, capping, and stabilization. First, deprotection removes the protection groups. Second, during coupling the 5' to 3' linkage is generated with the incoming nucleotide. Next, a capping reaction is completed to prevent uncoupled nucleotides from forming unwanted byproducts. Lastly, stabilization is achieved through an oxidation reaction that makes the phosphate group pentavalent. For a more detailed description of this process, read this article from IDT DNA.

Protocols

Part 1: Research pCTCON2 expression vector

In order to study the effects of cell surface display of your peptide of interest in yeast, a plasmid vector must be used to introduce your peptide into the yeast model system. The vector backbone includes several key features that enable successful expression of the peptides. To understand our model system, first familiarize yourself with the important features of the expression plasmid.

In your laboratory notebook, complete the following:

In this exercise, you will explore the features present in the plasmid that are necessary to express the peptide sequence (see plasmid map below).

• Describe the purpose / role for each of the following features that are present in the pCTCON2 plasmid backbone. Please note: you many need to reference resources outside of the wiki!
  • T3 promoter
  • Aga2
  • HA and myc epitopes
  • AmpR
  • TRP1
• Our expression vector is known as a "shuttle vector". What is this term, and what features of our vector enable this performance?
• Your peptide will be inserted between two features on this map. Which ones? Why?

Part 2: Prepare YSD peptide oligos

The instructors took DNA sequence you selected for your display peptide and added flanking DNA sequences to the oligos so that we could orient the peptide with our detection tags.

The Forward primer sequence added was "5 - GGCGGATCCGAACAAAAG - 3" at the end of your sequence so that your final display peptide will include 3 flanking amino acids at the C terminus (Gly-Gly-Ser) which provide a spacer between your peptide and the C-terminal myc tag.

The Reverse primer sequence added was "5 - AGCCTGCAGAGCGTAG - 3" at the beginning of your sequence so that your final display peptide in include 3 flanking amino acids at the N terminus (Leu-Gln-Ala)which provide a spacer between your peptide and the N-terminal HA tag.

Adding these additional bases to create your primer allows the primers to become the right length to anneal correctly to the vector and also results in spacers between your peptide and the tags.

While you were away the sequences for the insertion primers you designed were submitted to Genewiz. Genewiz synthesized the DNA oligos then lyophilized (dried) it to a powder. Follow the steps below to resuspend your oligo (or 'primer').

1. Centrifuge the tubes containing your lyophilized oligos for 1 min.
2. Calculate the amount of water needed to give a stock concentration of 100 μM for each oligo.
3. Resuspend each primer stock in the appropriate volume of sterile water, vortex, and centrifuge.
4. Calculate the volume of your stock that is required to prepare a 20 μL of solution that contains your mutagenesis oligo at a concentration of 10 μM.
   • Try the calculation on your own first. If you get stuck, ask the teaching faculty for help.
5. Prepare a primer mix that contains both your forward and reverse oligos at a final concentration of 10 μM in 20 μL of sterile water.
   • Be sure to change tips between primers!
6. Return the rest of your peptide insertion oligo stocks, plus your primer specification sheet, to the front bench.

Part 3: Use site directed mutagenesis to introduce your peptide sequence into pCTCON2

To perform site-directed mutagenesis (SDM), custom designed oligonucleotides, or primers, are used to incorporate mutations into double-stranded DNA plasmid as a specific location. These mutations can change the bases of the sequence, delete bases, or insert bases. One approach to SDM is to use primers that align to the sequence in the plasmid in a back-to-back orientation. As shown in top left of the schematic below, the primers (forward primer = black arrow and reverse primer = red arrow) anneal to the plasmid such that the 5' ends of the primers anneal to the DNA in a back-to-back orientation. In Step #1 of the schematic, the forward primer is used to replicate the top strand (outside circle of the plasmid) and the reverse primer is used to replicate the bottom strand (inside circle of the plasmid). The resulting single-stranded products (extension from each primer generates a single-stranded product) are able to anneal due to sequence homology, as shown in the first quadrant of the zoom-in for Step #2. In Step #2A the 5' ends of the linear, single-stranded amplification products are phosphorylated to prepare for ligation (Step #2B). Remember that a 5' phosphate is required for 3' OH nucleophilic attack, this results in circular plasmids.

Thus far in this description of SDM, one very important detail has not been mentioned. How specifically is the insertion coded in the primers incorporated into the plasmid sequence? In the top left of the schematic, the forward primer contains a "squiggle" mark that represents the desired insertion. The single-stranded product that results from extension from this primer will contain the desired insertion and therefore be incorporated into the products generated in Step #1. Lastly, in Step #2C the plasmid template that contains the unmutated parental sequence is destroyed so that only the plasmids with the desired insertion are present at the end of the procedure.

For this procedure we are using the Q5 Site Directed Mutagenesis Kit from NEB. A more technical depiction of the protocol you will use to introduce a peptide sequence insertion is included below. Briefly, in Step 1 DNA polymerase copies the plasmid using the forward primer to insert the new DNA sequence. Following PCR amplification the product is a linear DNA fragment. In Step 2 circular plasmids that carry the point mutation are generated when the double-stranded DNA is phosphorylated (Step 2A) and then ligated (Step 2B). Following the amplification reaction, the expression plasmid template that does not contain insert is present in the reaction product. To ensure that only the insertion-containing expression plasmid is used in the next steps, the parental DNA is selectively digested using the DpnI enzyme (Step 2C). The underlying selective property is that DpnI only digests methylated DNA. Because DNA is methylated during replication in host cells, DNA that is synthetically made via an amplification reaction using PCR is not methylated. Lastly, in Step 3 the insert-containing expression plasmid is transformed into competent cells that propagate the plasmid.

Each group will set up one reaction. You should work quickly but carefully, and keep your tube in a chilled container at all times. Please return shared reagents to the ice bucket(s) from which you took them as soon as you are done with each one.

1. Retrieve one PCR tube from the front laboratory bench and label the top with your team color and lab section (write small!).
2. Add 10.25 μL of nuclease-free water.
3. Add 1.25 μL of your primer mix (each primer should be at a concentration of 10 μM).
4. Add 1 μL of CTCON2 plasmid DNA (concentration of 25 ng/μL).
5. Lastly, use a filter tip to add 12.5 μL of Q5 Hot Start High-Fidelity 2X Master Mix - containing buffer, dNTPs, and polymerase - to your tube.
6. Once all groups are ready, we will begin the thermocycler, under the following conditions:

Segment	Cycles	Temperature	Time
Initial denaturation	1	98 °C	30 s
Amplification	25	98 °C	10 s
		63 °C	30 s
		72 °C	3 min
Final extension	1	72 °C	2 min
Hold	1	4 °C	indefinite

After the cycling is completed, you will complete the KLD reaction (which stands for "kinase, ligase, DpnI").

1. Add the following reagents:
   • 1 μL of your amplification product
   • 5 μL 2X KLD Reaction Buffer
   • 1 μL KLD Enzyme Mix
   • 3 μL nuclease-free water
2. Incubate the reaction for 5 min at room temperature.
3. Then, use 5 μL of the KLD reaction product to complete a transformation into an E. coli strain (NEB 5α cells of genotype fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17).
   • The transformed cells will amplify the plasmid such that you are able to confirm the appropriate mutation was incorporated.
4. Transform the cells using the following procedure:
   • Add 5 μL of KLD mix to 50 μL of chemically-competent NEB 5α.
   • Incubate on ice for 30 min.
   • Heat shock at 42 °C for 30 s.
   • Incubate on ice for 5 min.
   • Add 950 μL SOC and gently shake at 37 °C for 30 min.
   • Spread 150 μL onto LB+Amp plate and incubate overnight at 37 °C.

Reagents list

• pCTCON2 vector (a gift from the Wittrup lab)
• Q5 Site Directed Mutagenesis Kit (from NEB)
  • Q5 Hot Start High-Fidelity 2X Master Mix: propriety mix of Q5 Hot Start High-Fidelity DNA Polymerase, buffer, dNTPs, and Mg²⁺
  • 2X KLD Reaction Buffer
  • 10X KLD Enzyme Mix: proprietary mix of kinase, ligase, and DpnI enzymes
• SOC medium: 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose
• LB+Amp plates
  • Luria-Bertani (LB) broth: 1% tryptone, 0.5% yeast extract, and 1% NaCl
  • Plates prepared by adding 1.5% agar and 100 μg/mL ampicillin (Amp) to LB

Navigation links

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