Difference between revisions of "20.109(F18):Generate gRNA plasmid (Day3)"

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(Part 2: gRNA oligo preparation)
(Part 3: Complete gRNA insertion and amplification reaction)
 
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*After the cycling is completed, the teaching faculty will complete the KLD reaction (which stands for "kinase, ligase, ''Dpn''I") using 1 &mu;L of your amplification product, 5 &mu;L 2X KLD Reaction Buffer, 1 &mu;L KLD Enzyme Mix, and 3 &mu;L nuclease-free water. The reactions will be incubated for 5 min at room temperature.
+
*After the cycling is completed, you will complete the KLD reaction (which stands for "kinase, ligase, ''Dpn''I") using  
 +
**1 &mu;L of your amplification product
 +
**5 &mu;L 2X KLD Reaction Buffer
 +
**1 &mu;L KLD Enzyme Mix, and  
 +
**3 &mu;L nuclease-free water.  
 +
*Incubate the reaction for 5 min at room temperature.
  
*The teaching faculty will then use 5 &mu;L of the KLD reaction product to complete a transformation into an ''E. coli'' strain (NEB 5&alpha; cells of genotype ''fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17'') that will amplify the plasmid such that you are able to confirm the appropriate insertion (or 'mutation') was incorporated.  The transformation procedure will be as follows:
+
*Then, use 5 &mu;L of the KLD reaction product to complete a transformation into an ''E. coli'' strain (NEB 5&alpha; cells of genotype ''fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17'') that will amplify the plasmid such that you are able to confirm the appropriate insertion (or 'mutation') was incorporated.  The transformation procedure will be as follows:
 
#Add 5 &mu;L of KLD mix to 50 &mu;L of chemically-competent NEB 5&alpha;.
 
#Add 5 &mu;L of KLD mix to 50 &mu;L of chemically-competent NEB 5&alpha;.
 
#Incubate on ice for 30 min.
 
#Incubate on ice for 30 min.

Latest revision as of 20:11, 15 October 2018

20.109(F18): Laboratory Fundamentals of Biological Engineering

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Fall 2018 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Measuring genomic instability        2. Modulating metabolism        3. Engineering biomaterials              


Introduction

During the previous laboratory session, you designed a gRNA that will be used to target the E. coli fermentation pathway such that either ethanol or lactate production is increased. Your task for today is to ‘insert’ the gRNA sequence into an expression vector. Before we continue, we should think more about how the sequences you designed were used to generate actual primers that can be used to amplify and, in SDM (site-directed mutagenesis), incorporate mutations in DNA. Current oligonucleotide (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 detailed description of this process, read this article from IDT DNA.

We will use a cloning technique referred to as site-directed mutagenesis (SDM) to include your gRNA target sequence into the pgRNA expression vector, which will enable transcription of your gRNA. Following transcription, the gRNA molecule will bind to the E. coli genome at the complementary sequence (this is the target sequence you selected). Upon binding, the dCas9 protein will be recruited and will prevent RNA polymerase from transcribing the targeted gene.

Schematic of CRISPRi preventing transcription by RNA polymerase. Image taken from Qi Lab Homepage.

Unlike more commonly used CRISPR-based technologies, CRISPRi is used to modulate expression from the genome rather than to modify the genome. This distinction is due to the use of an enzymatically-inactive dCas9 (or ‘dead’ Cas9) protein. Because dCas9 is enzymatically inactive, it is unable to cleave the DNA upon binding to a gRNA/DNA complex. The lack of DNA cleavage results in gene silencing through impeding RNA polymerase binding, transcription factor binding, and/or transcription elongation. The method of repression is largely due to the location of the genome that is targeted. As you may recall from your gRNA design considerations, if you target the promoter of a gene of interest, you can influence transcription by all methods, and if you target a sequence within the gene, you will influence only elongation.

Protocols

Part 1: BE Communication Lab workshop

Our communication instructors, Dr. Sean Clarke and Dr. Prerna Bhargava, will join us today for a workshop on preparing and delivering your Journal Club presentation.

Part 2: gRNA oligo preparation

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

  1. Centrifuge the tube containing your lyophilized gRNA oligo for 1 min.
  2. Calculate the amount of water needed to give a stock concentration of 100 μM.
  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 100 μL of solution that contains your gRNA 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. Recall from the M2D1 exercise that an amplification reaction requires two primers. The gRNA oligo will serve as the forward primer in the insertion and amplification reaction and a universal CRISPRi oligo will be the reverse primer.
    • Obtain an aliquot of the CRISPRi reverse primer (resuspended at a concentration of 100 μM) from the front bench.
  6. Prepare a primer mix that contains both your gRNA oligo (or 'primer') and the CRISPRi reverse primer at a final concentration of 10 μM in 100 μL of sterile water.
    • Be sure to change tips between primers!
  7. Return the rest of your gRNA oligo stock, plus your primer specification sheet, to the front bench.

Part 3: Complete gRNA insertion and amplification reaction

We will be using the Q5 Site Directed Mutagenesis Kit from NEB to insert the gRNA sequence into an expression vector. For this procedure you will combine the gRNA primer you designed, a universal CRISPRi primer specific to pgRNA, and the pgRNA plasmid DNA encoding. DNA polymerase will copy the plasmid using the gRNA primer to insert the target sequence you selected. Following this reaction the 'mutated' product is a linear DNA fragment. To generate circular plasmids that carry the gRNA sequence, the DNA is phosphorylated then ligated. In addition, there is still parental -- that is, non-mutant -- DNA present in your reaction product. To ensure that only the gRNA-containing plasmid is used in the next steps, the parental DNA is selectively digested using the DpnI enzyme. The underlying selective property is that DpnI only digests methylated DNA. Therefore, the synthetically made (and thus non-methylated) mutant DNA is not digested, while the parental DNA is digested due to methylation by the host bacterial strain originally used to amplify it. The resulting small linear pieces of parental DNA are simply degraded by the bacteria upon transformation, whereas the intact (due to the phosphorylation and ligation reaction) circular mutant DNA is amplified by the bacteria.

Generating insertions using SDM technique schematic. Image modified from Q5 Site-Directed Mutagenesis Kit Manual published by NEB.

Each group will set up one reaction, for your insertion. Meanwhile, the teaching faculty will set up a single positive control reaction, to ensure that all the reagents are working properly. 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. Get a PCR tube 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 pgRNA 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
55 °C 30 s
72 °C 2 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") using
    • 1 μL of your amplification product
    • 5 μL 2X KLD Reaction Buffer
    • 1 μL KLD Enzyme Mix, and
    • 3 μL nuclease-free water.
  • Incubate the reaction for 5 min at room temperature.
  • 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) that will amplify the plasmid such that you are able to confirm the appropriate insertion (or 'mutation') was incorporated. The transformation procedure will be as follows:
  1. Add 5 μL of KLD mix to 50 μL of chemically-competent NEB 5α.
  2. Incubate on ice for 30 min.
  3. Heat shock at 42 °C for 30 s.
  4. Incubate on ice for 5 min.
  5. Add 950 μL SOC and gently shake at 37 °C for 1 h.
  6. Spread 50 μL onto LB+Amp plate and incubate overnight at 37 °C.

Reagents

  • 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+.
    • Universal CRIPSRi reverse primer 5' - ACT AGT ATT ATA CCT AGG ACT GAG CTA GC - 3'
    • 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 contains 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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