Introduction

In the next few chapters we will go through several alternative cloning strategies. These each have advantages and disadvantages which will be briefly discussed.

As you may recall from the section on CRISPR, we often utilize naturally occurring processes from organisms like bacteria and viruses – and modify them for our use. Gateway cloning is another example of this. We will begin by briefly describing the natural process we are borrowing, followed by a description of how it has been altered for cloning. Finally we will cover the procedures of insert generation, the production of an entry clone and the selection process. We’ll touch on making a destination vector – you may get some practice designing one in the final bioinformatics assignment.

A. How Lambda phage DNA is inserted into E. coli DNA:

Bacteriophages can infect bacteria using two different modes. In the first, the phage’s genome is inserted into the bacterium where it is transcribed and translated by the host’s proteins. The result is that the bacterium is converted into a phage factory, producing a huge number of new phage particles. Eventually the cell bursts open and distributes the phages into the environment, where they can infect additional bacteria. This mode is called lysis or the lytic phase.

However there is a second mode of infection that could occur. In this situation, the phage DNA is inserted into the bacterial genome where it stays for potentially many generations. When the bacterial genome is replicated, the viral genes are replicated too and passed to the next generation as the cell divides. This is called lysogeny. The phage genome (called the prophage) can remain stably in the bacterial chromosome for a long time but under certain circumstances, such as UV light or some other types of stress, the phage DNA can excise from the bacterial chromosome. At this point, the lytic cycle takes place.

The phage insertion into the bacterial chromosome is an example of site-specific recombination (crossover). These require specific sequences in the phage genome and the bacterial genome. The sequences pair and the crossover event is catalyzed by proteins that recognize and bind to the sequences.

Click here to download the powerpoint slides presented in video below.

A-1. The attB  and attP sites

The recognition sequence in the bacterial genome is found in just one location, between the bio and gal operons. The sequence is called attB (att is short for attachment and the B means bacterial) It is 30 bp long and contains a 15 bp core where the crossover takes place.  The site to the left of the core is called B and the site to the right of it is called B’. Together the sequence is named: BOB’. The O refers to the core.

The corresponding sequence in the phage DNA is called attP (att means attachment and P means phage). It contains a 15 bp core that is identical to the attB site. To the left of the core is the P sequence and to the right of it is the P’ sequence. Together they are called: POP’. The attP site is more complex and longer than the attB site. Though the sequences of P and B, and P’ and B’ have some similarities they are not identical except for the core sequence.

These sites can align with each other and then a restriction enzyme makes a sequence specific cut and the pieces are ligated together. If the same pieces that were cut join together, the restriction enzyme cuts it again, but if the different pieces are joined (attB with attP) then the restriction recognition site is lost. This should sound a bit familiar – it is similar to why we included EcoRV into our blunt end ligation in our first cloning project. The result is that these enzymes catalyze a single crossover between the circular Lambda phage genome and the circular E. coli chromosome. This generates a single larger circle which now contains the phage genome inside the bacterial genome. The crossover occurs inside the O sequence that is common to both sites. Two new sites are generated to either side of the phage genome. On the left is a site composed of BOP’, called attL and on the right is a site composed of POB’ called attR.

The phage DNA can excise back out of the bacterial chromosome through reversing the recombination event, with the attL and attR sequences aligning and a crossover event that regenerates the original att B and att P sequences. The enzyme mixes that catalyze the insertion of the phage genome and those that catalyze its excision differ by one enzyme, which is required to perform the reverse crossover reaction that will excise the phage genome. It recognizes the attL and attR target sequence and specifically cuts that sequence. When the cut ends are joined together, if they generate attL and attR sequences again the enzyme cuts them again. If they generate the attB and attP sequences, the reaction stops.

Click here to download the powerpoint slides presented in video below.

B. Co-opting the site-specific recombination system:

B-1. Using site specific recombination for cloning

You can imagine, I hope, that when we understand how a site specific recombination system works, it gives genetic engineers something interesting to think about. Since crossover occurs at a specific recognition sequence, can we take those recognition sequences use them to join two – or more – DNA sequences together? The key to being able to do this is being able to attach the appropriate recognition sequences to the DNA molecules we want to join.

What kind of system would we design?

It would not (necessarily) require the use of restriction enzymes or ligase. Cloning that uses these is not very efficient. If we used a site specific recombination system, we would expect higher efficiency – so more correct clones in a reaction.

We would also want to be able to potentially join multiple DNA molecules in a single reaction and have some means of selecting the clones we want very accurately. Blue-white selection is not an option in gateway cloning – or if not impossible it would certainly be very complicated. Think about why.

B-2. Gateway cloning

In the Gateway cloning system, att sequences are needed to either side of the insert we want to introduce into a vector.  So, the attB and attP sequences were slightly redesigned to produce attB1 and attB2 that could cross over with attP1 and attP2 sequences, respectively. This allows for directional cloning – if you place the recognition sequences appropriately you should only get clones with the insert in the correct location and orientation.

B-3. Making the entry clone

To start in Gateway cloning, you insert the gene of interest into an entry vector. This vector, containing your insert is called an entry clone. Your DNA can be transferred from the entry clone into multiple additional vectors, called destination vectors, very quickly and relatively easily.

A common procedure for starting gateway cloning involves incorporating attB1 and attB2 sites into primers used to PCR amplify your insert.  This is followed by a BP clonase reaction to insert your DNA into the vector, which is called pDONR.  However, you can also use regular restriction enzyme cloning to put your first insert into a gateway vector. In this case, the vector has attL sites (i.e. attL1, attL2) to either side of the cloning site.

And you can also use topoisomerase cloning. Topoisomerases relieve supercoiling that occurs during replication or transcription, by cutting the DNA strand and then re-ligating it after the supercoils unwind to relieve strain on the molecule.  There are vectors that have been made with topoisomerase attached to the cleaved ends of the vector at the cloning site. There is a T overhang on the vector and our PCR product with its complementary A overhang fits into the vector and is ligated by the topoisomerase, which is removed from the vector during this process. There are many types of vectors that use Topoisomerase cloning and some of these are designed for use in Gateway cloning. These also have attL sites to either side of the insertion point.

The image below outlines the PCR method you can use to make the entry clone with your GOI (gene of interest, or other sequence of interest) in it.  In the image, the GOI is shown in blue with the blue arrows indicating forward (left) and reverse (right) primers. The green and red squiggles on the 5′ ends of the primers indicate the extra sequence added to the primers:  attB1 (green) and attB2 (red).  The PCR product generated then has the GOI with the att sequences to either side of it.  This product can be combined with the pDONR (or other) vector, which has att P1 and att P2 sites on it.  In the BP clonase reaction, the vector that has undergone recombination now contains the GOI which has “swapped in” while the linear PCR piece now has a segment from the donor vector “swapped in”. The linear piece has att R1 and R2 sites on it and the vector, now called the entry clone, has att L1 and L2 sites to either side of the GOI fragment.  The clonase reactions are very efficient, so the majority of plasmids will have undergone recombination, but some of the original vector and the original PCR fragment may be left in the tube. The linear pieces are not a problem because these cannot transform bacteria, but we want to ensure that the plasmids that transform bacteria are the correct ones. We’ll talk about how we can select the correct transformed cells later in this section.

Click here to download the powerpoint slides presented in video below.

B-4. Transferring your insert into destination vectors

Gateway cloning involves putting your insert into an entry vector. One vector for this that we’ve used in past offerings of 357 is called pDONR.  From this construct you can then transfer the insert to many other vectors for a multitude of purposes, such as RNAi constructs, reporter constructs, over-expression constructs etc. Over-expression constructs haven’t really been discussed, but generally they are constructs in which your gene is connected to a strong ubiquitous promoter in a construct which – once introduced into the organism or cells – will cause stronger expression than is usual for the gene. Sometimes an over-expression phenotype is no different from wild type but sometimes it is very informative. It depends on the function of  gene you are studying.

Advance planning is necessary for determining which att sites flank your insert at each step of the cloning.  You usually start with the att sites on the destination vector you want to use and then work backwards to determine which entry vector you should use to transfer the insert and then before that, what att sites to add to the primers for amplifying the insert. And, you can also use other methods to get the initial insert into the entry plasmid, as already mentioned.

B-5. Selection for the insert and vector you want

In Gateway cloning we make an entry vector with our insert but we usually want to move that insert from one plasmid to another for a specific purpose, as noted above. So in the gateway “reaction” tube,  we put the entry clone we made (e.g. pDONR plus our GOI) with the attL1 and L2 sequences to either side of it, and the destination vector, a plasmid with the correct att  (R1 and R2, e.g.) sites to crossover with the entry vector and an insert between those.  We want to swap inserts between the two plasmids in the reaction. So in the tube, we will have two plasmids and two inserts. And although the crossover reaction is quite efficient we would still expect to have 4 different constructs in the tube after the crossover reaction: the original vector and either of the inserts, and the destination vector and either of the inserts. We need to be able to select for the one combination we want – your gene of interest in the destination vector – and against the ones we don’t want – all the others.

We use two methods to do the selection. The first is that the two vectors should have differing antibiotic resistance. So if the entry plasmid was resistant to ampicillin, but not kanamycin and the destination vector was resistant to kanamycin but not ampicillin, then we plate our transformed cells on kanamycin. The entry plasmid with either the gene of interest or the insert swapped in from the destination vector won’t allow transformed cells to grow on these plates. So now we have just two plasmids to worry about. One is the destination vector with the GOI insert. That is the one we want. But there will also be some destination vectors with their original insert in the tube. These will be vectors that did not undergo the intended crossover and so don’t have the GOI.  How can we distinguish between these?

It is the insert from the destination vector that is the key here. The insert contains the ccdB gene. This gene encodes a protein that interferes with DNA replication (it inhibits gyrase) and so bacteria that have a plasmid with this gene on it will not be able to replicate their DNA and will thus not be able to divide and grow into a colony on the kanamycin plate. When you read about this you will see that it is sometimes referred to as the “cell suicide” gene, which I think is kind of insensitive and it isn’t really accurate either. It is more like a cell death gene.

You may wonder how we could ever grow and maintain a plasmid that has the ccdB gene on it in cells. We can’t do so in normal cells; we have to use a special strain that has a mutation in the gyrase gene that prevents the ccdB gene product from interfering with DNA replication.  The image below is an outline of the LR clonase reaction needed to move the GOI from your entry vector into the destination vector.  Look at the figure and determine how we select against the entry vector (with either insert) and against the destination vector that does not have our desired insert. Note that “entry” and “destination” are general terms. In a cloning experiment you would use the correct names of the plasmids.

B-6. Assembling multiple fragments into a single construct:

Since the beginning of Gateway technology, many variant att sites have been designed. There are att B1, B2, B3, B4 etc. and att P sites that match these. There are multiple attL sites also, L1, L2, L3  and the corresponding attR sites, etc. that are formed when the relevant B and P sites undergo crossover. It is possible to assemble several  pieces of DNA together in a particular orientation in just one reaction by ensuring that you have all the att sites matched up.  There are websites that will help you design the primers and will do a “virtual” cloning experiment in advance to check that your design will work. Keep in mind that each att site has just one corresponding site it can crossover with, so attB1 will only crossover with attP1, and attL3 can only crossover with attR3, and so on. Each B site needs a corresponding P site and each R site needs a corresponding L site.

The websites are helpful in another way too. The addition of the fairly long att sites to an insert can be challenging if you intend to express a protein. Reading frame matters for expression constructs and so the website will help you determine whether your insert is in frame for the expression you intend. One value of then using additional att sites is that if your insert is in frame in the entry vector, it will be also in the destination vector – the value of good design from the manufacturers!

Click here to download the powerpoint slides presented in video below.

Resources:

This site has all the basics for understanding Gateway cloning. It does not cover assembling multiple fragments as will be done in the simulation, though, so make sure to consult the theory pages of the simulation.

Plasmids 101: Gateway Cloning

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