Ligate Sticky Ends via DNA Ligation

DNA Ligation of Sticky Ends

Ligation of Sticky Ends, Summary of DNA ligation

We have already discussed a high level view of gene cloning in our Molecular Cloning Guide blog post. However, in that blog post we didn’t delve very deep into how we can perform each of the individual steps. Today’s blog post is about ligation. Ligation is the process by which two pieces of DNA can be glued together to form one piece. So, to begin, let’s assume you’ve already decided on a gene product that you want to clone. You’ve also designed primers and completed PCR on the open reading frame in your donor DNA (this could be genomic or non genomic DNA). Your next steps are to digest the PCR product with restriction enzymes and generate sticky ends. You’ll also want to digest your “shuttle” plasmid to generate complimentary sticky ends which will allow your “insert” DNA to click into position into your vector. It’s like a puzzle piece!

Note: It might be useful to look at our RNA Extraction & Isolation guide if you’re planning on making cDNA related to your gene

The above summary is demonstrated here:
Ligate Sticky ends using Ligase

Sticky Ends Insert into a Shuttle Vector

Only some Restriction Enzymes Create Sticky Ends

As you can figure out, generating sticky ends and complimentary ends is extremely important to the above process. However, several different restriction enzymes are available and each of them has different locations where they cut. Also, the type of cuts that they introduce may be “sticky” or “blunt”. Depending on the cloning strategy you are using, you may mix and match different enzymes to achieve different end goals. Ligation of “sticky ends” is much more efficient than ligation of “blunt” ends. Typically 10-100 times more T4 Ligase is required for blunt ends.

Here’s an image with various restriction enzymes and the kinds of ends they produce. Depending on the type of ends, your DNA ligation will proceed very differently!

Restriction Enzymes for DNA Ligation

Ligate DNA via DNA Ligase

Once the restriction enzyme digestion is complete, you can proceed to the ligation step. But, before you digest anything, make sure you’ve planned everything properly! You need to make sure that the insert will be ligated in the proper direction in the shuttle vector. Only once you’ve vetted your overall strategy, should you proceed to ligation and transformation, etc.

There are several kinds of ligase enzymes but the enzyme produced by T4 bacteriophage-infected E. Coli is the most common one. This ligase is called T4 ligase. Whereas normal E. Coli produce DNA ligase that uses NADH as a cofactor, T4 infected E.Coli produce a ligase that uses ATP as a cofactor. This enzyme will find the 3′ Hydroxyl and 5′ Phosphate within your sticky ends and it will form a phosphodiester linkage. If this is confusing, check out the Polymerase chain reaction (PCR) guide for images on what DNA looks like. This is shown here:

Ligation Protocol for T4 Ligase

Phosphodiester Bond Formation during Ligation

Protocol for Ligation of Transgene Insert into Shuttle Vector

Ligation enables fragments of DNA to be combined, such as the cut ends of transgene inserts and plasmids during cloning. This protocol describes the directional cloning of a XbaI/SalI-digested transgene into a shuttle vector, pAdtrackCMV, via cohesive end ligation.

Materials for DNA Ligation

XbaI/SalI digested, gel-purified insert (approx. 1 kb) and pAdTrack-CMV shuttle vector (approx. 9.3 kb; Plasmid #16405, Addgene)
Quick Ligation Kit (contains DNA ligase and 2X Reaction Buffer; #M2200S, New England Biolabs)
Agarose plate containing ethidium bromide
DNA standards

Ligation Methodology

  1. Estimate the DNA concentration of purified insert and vector preparations by applying 1 µl to an agarose gel plate (+ethidium bromide) alongside a range of DNA standards and visualizing under UV light.
  2. Prepare the ligation mix as follows:

    XbaI/SalI digested pAdtrackCMV 50 ng

    XbaI/SalI digested insert 17 ng

    Add water up to 10 µl total volume.
  3. Add 10 µl of 2X Reaction Buffer and mix.
  4. Add 1 µl of DNA ligase and mix.
  5. Microcentrifuge briefly to settle liquid to the bottom of the tube and incubate at 25°C for 5 min.
  6. Place on ice* and transform into desired bacterial strain.

Tips and Tricks for DNA Ligation

  • This reaction setup is using a digested insert to vector DNA molar ratio of 3:1. Inserts of different sizes will require a different amount to be added. Important ligation control reactions to include are (1) digested vector only and (2) digested insert only.
  • Ligation reactions can be stored at -20°C for future use

Applications of Ligation on SciGine

Construction of PB42 Vectors Via Ligation
Plasmid Construction via PCR and Ligation
Plasmid Ligation and Transformation in Yeast
Construct with Human p275UTR
Different DNA ligation methods discussed

Video Tutorial About Sticky Ends and Ligation

References

He et al Proc Natl Acad Sci U S A. 1998 Mar 3. 95(5):2509-14.
Sticky Ends Explained Well
DNA Ligation Theory
Gaastra et al. Ligation of DNA via T4 Ligase
Tsuge et al. One Step Assembly of DNA fragments

Protein Purification of Recombinant Proteins

Protein Purification of Recombinant Proteins

Protein Purification Summary

In our previous blog posts we have explored Gene cloning with Plasmid Vectors in Bacteria, Transient transfection into Mammalian Cells with Calcium Phosphate, and how we can use newly introduced proteins to control biology. Proteins made this way are considered recombinant because they aren’t natively produced in the organism that you got them from.  We really like recombinant technology because it allows us to scale up protein production and generate therapeutic and/or interesting fusion proteins that we can use. If you want some human protein, would you rather grow humans and isolate the protein for scale up (~30 years per doubling)? Or use bacteria instead (~20 minutes per doubling)? Note: this was a joke. Don’t grow humans for protein production 🙂

In this blog post, we are going to explore how “recombinant” proteins can be purified after cells have expressed the gene products that you cloned into them. The strategies explored here can be applied to all sorts of proteins so let’s begin!

Protein Purification of therapeutic recombinant proteins

Strategies for Protein Purification

Let’s say you have some bacteria that you’ve produced a protein inside. Your first step is to lyse those bacteria and neutralize any proteases that are now in your lysate. Proteases will wreak havoc on all the proteins in solution…so this step is important. Next, we have to think about the recombinant protein that we created in order to purify it. Several different purification methods can be used based on your properties:

    • Protein Charge: If your protein has a overall charge because of excess arginine or aspartamine residues, perhaps it can be purified by running it through an ion exchange column. For negatively charged proteins, use anion exchange chromatography, and for positively charged proteins use cation exchange chromatography. The steps here are simple…Dissolve your protein in a buffer and incubate it with the resin. Wash the resin with some low salt buffer. And then elute the bound protein with some high salt buffer (which breaks the ionic interactions with the resin).

Using Ion Exchange columns for protein purification

    • Protein Size: Dialysis and Size Exclusion chromatography can help you isolate proteins based on their size. In the case of dialysis, you incubate your protein in a dialysis bag and stir it while replacing the buffer outside. Your protein and larger proteins are retained in the bag while smaller proteins are filtered out through diffusion. Size exclusion chromatography (SEC) works similarly to separate out larger molecules from smaller ones. Take a look at our HPLC Step by Step guide to understand chromatography in general.

Recombinant proteins separated by size via dialysis

    • Protein Affinity: If you are lucky enough to buy resin with antibodies vs. your protein, you can simply pass your protein through the resin and it will selectively bind your protein. Then wash it a little bit with buffer so no other proteins are bound and finally elute it by disrupting the antibody-protein interaction.

Affinity based separation of recombinant proteins

SciPrice, biology product search engine

    • Protein Substrate: If your protein is an enzyme with a binding pocket, you can also immobilize your substrate on a column and use that for purifying your protein. Simply pass your protein through the column multiple times so it binds the substrate while other non-functional proteins are easily washed away.

Substrate affinity column for Protein purification

A typical protein purification strategy will involve using several of these techniques in combination. No single technique is 100% efficient, so each time you purify with one of these methods, your protein will get more and more pure. Use a western blot to analyze how clean your protein is. You can also use a silver stain to determine purity. I’ll discuss this technique in the future.

Purification of Recombinant Proteins with His Tags

Above, we have already discussed the purification of recombinant proteins via their charge and using their binding pocket. Another strategy that’s very popular is to introduce at least 6 Histidine residues into the N- or C-Terminus of a protein via cloning. Then, when it’s time for purification we can run the protein through a divalent nickel column. Histidine residues, at a high pH (~7.6), can chelate Nickel and hence will be bound on the nickel column. The column can be washed with a low concentration of Imidazole (~20 mM) and then eluted with 150 mM+ of Imidazole.

Cloned His Tags easily chelate nickel for separating recombinant proteins

Step by Step Guide to Purification of a His-Tagged Fusion Protein

Materials

Neurospora culture
Lysis Buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 1 mM B-mercaptoethanol)
Homogenizer
Protease inhibitor cocktail
Phosphate buffered saline (pH 7.0)
Wash buffer (50 mM Phosphate Buffer pH 7.0, 300 mM NaCl, 1 mM Imidizole, pH 7.0 final)
Elution buffer (50 mM Phosphate Buffer pH 7.0, 300 mM NaCl, 150 mM Imidizole, pH 7.0 final)
Collection tubes for washes and elutions

Methods

  • Grow culture and lyse in lysis buffer at 4 C for 45 min
  • Homogenize lysate and centrifuge at 12000 g for 20 min
  • Discard the pellet
  • Dialyze the supernatant against PBS (pH 7.0) for 1 hour at 4 C. Replace the buffer outside the dialysis bag and continue to dialyze for 1 hour more.
  • Prepare the Nickel-Agarose column according to the manufacturers instructions.
  • Add in your protein dialysate from the previous steps on top of the column.
  • Allow the material to diffuse to the bottom and load the filtrate on the column once again
  • Wash the column with wash buffer (use 10x the volume of the beads in the column)
  • Elute the column with elution buffer (use 1-3x the volume of the beads in the column)
  • Collect the eluant in 1 ml fractions and assay each fraction for protein
  • Assaying the protein can be performed via a western blot or other protein assay

Tips and Tricks for Purifying Recombinant Proteins with His Tags

  • EDTA is used in lysis buffer to prevent protease activity
  • Use a dialysis membrane of the appropriate size to retain your protein’s molecular weight + 1000 Da at least. This way you can be sure that you aren’t losing a lot of your protein along with all the filtrate.
  • The size of the column that you use should be determined according to the instructions
  • Protein assays for determining activity are a broad category. For many enzymes  there are assays where the enzyme will be used to cleave a substrate and generate a fluorescent signal.

Protein Purification Protocols on Scigine

Powerpoint related to various Purification Processes

References:

Guide to Protein Purification and Assays from NIH
Protein Purification Powerpoint Presentation
Applications of Protein Purification from Cornell
Manju Kapoor’s Guide to Protein Purification
Nickel-Agarose Purification Guide

Gene Cloning using Plasmids: Molecular Cloning Intro

Gene Cloning using Plasmids via Molecular Cloning techniques

Gene Cloning with Plasmids: Summary

We all know that DNA is the basic building block of biology. So, how can we make use of DNA to change cell biology? Well, today’s blog post will focus on “gene cloning” — making plasmids (circular DNA strands) so that we can introduce them into bacteria using our previous bacterial transformation method. With a plasmid inside the bacteria, you can a) use bacteria to make copies of the plasmid, b) make new proteins with the transformed bacteria and c) do the same inside mammalian cells using the Calcium Phosphate transient transfection method that we developed earlier. With molecular cloning techniques, we can control biology and make cells do some really cool stuff! Note: this is an overview post and does not have a step-by-step protocol associated with it. I’ll tease apart the different steps in future blog posts.

Molecular Cloning of Plasmids: Primer Design

“Cloning” refers to the process of making a copy of a gene so that we can modify it and see what happens. Remember, if you modify genes, your cells start producing new proteins; these proteins could be therapeutic and/or give your cells some new skills. To start, you’ll probably want to review the PCR protocol & guide to remember how PCR works. Now, let’s say we have a gene that we want to clone already available. The next most important part of PCR based gene cloning is the primer…so to design a primer, we need the following:

  • Hybridization sequence: A series of bases that compliment the bases right before your “target gene” or gene of interest.
  • Leader sequence: A few extra bases for our restriction enzymes to make efficient cuts that don’t overlap with our gene of interest.
  • Restriction sites: Places that we will cut so that we can make the plasmid circular.

Take a look at this image to understand the above plasmid design:
Making primers for Gene Cloning PCR

Molecular cloning primer design

Gene cloning product

Be Careful Designing Plasmid Primers for Gene Cloning

Based on the above image, you can tell that if an enzyme’s restriction site is inside your gene of interest, you cannot use that restriction enzyme because you’ll cut your gene. Also, you’ll be putting this gene into a new plasmid. Make sure that the restriction enzyme you use is compatible with the “multiple cloning site” within this new plasmid. If you end up inserting this gene in random locations, the probability that this plasmid will be incorporated into the bacteria or expanded will be significantly decreased.

Look at the image below to understand these tips:
Gene cloning failure - wrong restriction enzyme

Primer mismatch -- Gene cloning error

Gene cloning with PCR

With the primer already designed, we are ready to clone our gene. The rest of the steps in the gene cloning process are:

  • PCR everything
  • Use restriction enzymes to digest the PCR product
  • Use Gel Electrophoresis to purify the insert and the “vector” (recipient plasmid)
  • Ligate the plasmid
  • Transform bacterial cells
  • Isolate our plasmid for future use
  • Analyze the PCR products

Since we already know how to do PCR from our previous blog post, let’s focus on the other stuff. The first step listed is to digest the PCR product. For this, we will use restriction enzymes and incubate them with the PCR products. If everything was designed properly, we would know exactly where the restriction enzymes will cut the DNA in both the “vector” and the “insert”. Next, we will run these restriction digests on a gel and pick out the bands corresponding to our vector and insert (which we already know the size of). Any other “junk” PCR products will be removed in this step. The vector and insert DNA will then be “ligated” to form our new plasmid. To confirm our gene is in this plasmid, we will transform some bacteria with it on a petri dish. Try to make dilutions of your bacteria so that you can grow colonies of bacteria and pick out colonies later on. With the colonies that you pick out, you’ll want to isolate their DNA and digest it to see if your vector and insert are inside. We’ve already isolated the vector and insert in the past, so it’s simple to find out if our insert is inside the bacteria. Finally, as another confirmation, we will sequence the DNA from the bacteria and confirm that everything exists. We will write more about each of these steps in the future, but we wanted you to see them together, as an overview, in this blog post.

Take a look at the steps below:

Preparing plasmid vectors for Gene Cloning

Electrophoresis and Ligation of Genes using Restriction Digests

Transformation of Bacteria and Isolation of Final Gene Clones

Tips and Tricks with this methodology

PCR

: Make sure you choose the melting temperature to match the part of the primer that binds the “open reading frame” (your gene of interest). If you choose the wrong melting temperature, you might get the wrong PCR products because either a) your Tm was too low and you didn’t split the ORF or b) your temp was too high and you got lots of non specific binding.

DNA Digestion

: Make sure DNA digestion occurs for a long time, preferable overnight, to make sure all your vector and insert products were cut and maximize your ligation in the next step. You may need to use alkaline phosphatase in this step. I’ll speak more about that in the future.

Gel electrophoresis

: During gel electrophoresis make sure that you run the correct controls and *know* what wells relate to each of the digested products. Also, make sure you skip lanes to make cutting the wells easier. After this, you’ll need to quantify your DNA so you have enough for the ligation step. You can use a UV spectrometer for this step.

Ligation

: Ligation also requires you to have several controls. For example, you need a ligation reaction without any insert. This will tell you how much background self-ligation your recipient plasmid has. You also need a ligation with some of the other bands you see during your gel electrophoresis. This will tell you how much contaminant DNA there was in your ligation.

Methods related to Gene Cloning on SciGine

Video about Gene Cloning with Plasmids

Notes from our audience

  • “TA cloning is another approach if cloning doesn’t work in systematic way” –Swapnil Oke on Linked In
  • “I think for completeness I think it would be valuable to also mention a few other plasmid features that are important. I didn’t see mention of ribosomal binding sites (RBS) or origin of replication, etc” – Michael Kim on Linked In. — I plan on write about more details regarding plasmid design and purification in the future. For now, please don’t use the above blog post as a comprehensive guide…more like an overview 🙂

References

Molecular Cloning book about PCR based cloning
Addgene Plasmid Reference, a comprehensive guide
Chaokun et al., Fast Cloning

Bacterial Transformation Protocol with Competent Cells

Bacterial Transformation with Competent Cells

Bacterial Transformation using Competent Cells: Summary

Since we have already learned Calcium Phosphate Transfection with mammalian cells, let’s now focus on bacterial transformation of DNA with competent cells. In general, bacterial cells take up naked DNA molecules or plasmids via a process called transformation. Usually, this happens at a slow rate, but when bacterial cells die in close proximity to others, or when they are stressed, the transformation process occurs at a much higher rate. However, not all bacterial cells can be transformed, so biologists use ‘Competent Cells’ which are more inclined to take up DNA. The end goal of transformation is to get bacteria that have your genes of interest so that they will replicate your genes along with their own. If the bacteria contain your genes of interest, you can use them to mass produce proteins, or just store them for extended periods of time because bacteria are so hardy. A good way to test whether your genes of interest were transformed is to include antibiotic resistance in your plasmid. This way, you can be fairly certain that if your bacteria are resistant to antibiotics, they are also carrying genes of interest to you.

Take a look at how natural transformation works:
Transformation Protocol with DNA

Transformation Biology in Bacteria

For bacteria, survival is key and transformation is one of their survival mechanisms. As biologists, we can make use of this survival mechanism for our benefit as well. To do this, we first incubate our competent bacteria with our plasmid and calcium chloride. Bacterial membranes are permeable to chloride ions, but not to calcium. So, as chloride ions enter the cell, the bacteria tend to swell (because they also intake water with chloride ions). Then we heat the bacteria in a process called ‘heat shock’ such that they turn on their survival genes. This causes the bacteria to uptake the surrounding plasmids. With the right design, this plasmid will then be recognized by bacterial DNA polymerases (remember our PCR Guide ?) and it will be expressed/replicated along with the bacteria’s normal DNA.

Take a look below to understand how biologists transform cells:

What is transformation

Transformation Biology

Selecting Transformed Bacteria with Antibiotic resistance in a plasmid

Selecting for Transformed Bacteria with the Lac Z Operon

Once your target plasmid is inside the bacteria, you still need to separate transformed cells from those that are not transformed. Another key challenge is that the transformation process may lead to some DNA being recombined so that your gene of interest is no longer functional. How do you select for cells that only contain functional target DNA that hasn’t been recombined? The trick is to use both antibiotic resistance and a Lac Z operon. By cloning your plasmid along with a Lac Z operon, you give your cells the ability to make a galactosidase protein. If cells have the galactosidase and you feed them X-Gal, they turn blue; cells without this operon are white. So, you first transform all your cells. Then you feed them IPTG to activate the Lac Z operon and cause cells to produce the galactosidase. Then you add in X-Gal and just pick out the bacteria that have functional Lac Z because the useful cells will be a bright blue!

Check out the figure below:

Transformation in Bacteria with LacZ

Transformation leads to Competent cells with LacZ operon

Bacterial Transformation Protocol

Transformation describes the uptake and incorporation of plasmid DNA into bacteria. Antibiotic resistance genes carried on plasmids allow selection of transformants. This protocol describes the transformation of DH5α E. coli with pAdtrackCMV (a vector carrying kanamycin resistance).

Materials for Bacterial Transformation

Ligation mix (20 µl) – insert ligated into pAdTrack-CMV shuttle vector (Plasmid #16405, Addgene)
DH5α competent cells (includes pUC19 DNA control; #18265017, ThermoFisher Scientific)
LB broth (#10855-021, ThermoFisher Scientific)
LB Agar selective plates (prepare from #22700025, ThermoFisher Scientific) with 50 µg/ml kanamycin (#15160054, ThermoFisher Scientific)

Step by Step Transformation Protocol

  1. Thaw competent cells on ice. Aliquot 50 µl into cooled Eppendorf tubes for each transformation reaction.*
  2. Add 5 µl of ligation mix to each tube.*
  3. Incubate on ice for 30 min.
  4. Heat-shock the cells for 20 sec in a 42°C waterbath.
  5. Place on ice for 2 min.
  6. Add 950 µl of warm LB broth per tube.
  7. Allow cells to recover at 37°C for 1 hour with gentle shaking.
  8. Spread 200 µl and 20 µl of each transformation mix onto warm selective plates.*
  9. Incubate plates overnight at 37°C.
  10.  

Notes on this methodology

  • We will talk about “Ligation” in another future blog post
  • Step 1. Unused cells can be refrozen and stored at -80°C for future use.
  • Step 2. As a transformation control, add 1 µl of pUC19 plasmid to one aliquot of cells (pUC19 confers resistance to ampicillin so will need to be seeded onto different selective plates).
  • Step 8. Transformation mix can be stored at 4°C and plated the next day if required.

Bacterial Transformation Video Tutorial

Applications of DNA Transformation on Scigine

References

Excellent Book about Bacterial Transformation
Guide to Common terms in Transformation – Oklahoma University
Compilation of History of Transformation and related protocols
He et al Proc Natl Acad Sci U S A. 1998 Mar 3. 95(5):2509-14.