1. In-silico cloning

Molecular cloning often means insertion of a gene or other DNA fragment into a genetic vector (usually a plasmid). This is equivalent to the construction of a recombinant DNA molecule.

Cloning is necessary in order to study the function of a gene, since the cloning change the context of the gene from its original environment.

Cloning is also almost always necessary in order to express the gene in a different organism, since controlling sequences such as promoters and terminators needed are different between organisms.

This document contains exercises that will show you how to make simple sequence manipulation on your computer and how to assemble the sequence of recombinant DNA molecules following a cloning strategy.

The main purpose of in-silico cloning is to predict the final sequence outcome of a cloning strategy or recombinant DNA construction project on the computer before performing the actual experiments in order to check the cloning strategy for errors.

The final sequence is also useful in order to find strategies to confirm a successful cloning.

You will practice:

• Clone a blunt DNA fragment in a vector digested with a blunt restriction enzyme
• Sticky end cloning using a restriction enzyme with:
  • 5' overhang
  • 3' overhang
  • Outside cutter

The DNA editor ApE is used for these exercises, but any other editor could be used. This document contains ten questions. Note your answers in a text file using Notepad or some other text editor.

Open a new empty window in ApE (Fig 2).

Copy the DNA sequence (black text) in Fig 3 and paste it into the ApE window.

This sequence represents a short gene consisting of a start codon, one amino acid (AAA = Ala) and a stop codon (***), but this is not important at this point. The CCCGGG are recognition sites for a restriction enzyme called SmaI. The sequence in Fig 3 represents the double stranded linear DNA molecule shown in Fig 4.

Now we need to find out how the enzyme SmaI cuts the DNA; we need to know which bonds are going to be broken by the enzyme. To do this go to Enzymes>Enzyme Selector in ApE and look at the SmaI enzyme (Fig 5).

Just put the cursor above the enzyme name as in Fig 5.

This information is also available from the restriction enzyme database.

Question 1: Does SmaI produce blunt cut or sticky ends?

Question 2: Use the same strategy to find out which ones of the following enzymes produce a blunt cut: BamHI, EcoRI, EcoRV, PvuII.

Fig 5 represents the text describing the cut SmaI makes depicted in Fig 6.

The ^ symbol in Fig 6 can be understood as a cut in each strand as depicted in Fig 7.

*

* If we digest our molecule with SmaI, we will obtain three molecules of double stranded DNA (Fig 8).

In order to find the SmaI site in the sequence, go to Enzymes>Enzyme Selector and click “Highlight” at the bottom of the page (Fig 9).

The resulting SmaI highlighting in the main sequence window can be seen in Fig 10.

Now open the pUC19 sequence in another ApE window, but keep the first sequence open as well. The pUC19 sequence can be found in the file: “plasmid_pUC19_in_raw_sequence_format_cSEGUID_n-NZfWfjHgA7wKoEBU6zfoXib_0.txt”.

This file should be present in the same folder as this document. Highlight the SmaI site in pUC19 in the same way as before (Fig 11).

Copy the 15 bp blue text in the middle from Fig 8 and paste it between the ccc and the ggg in the pUC19 sequence. It is important to take only the middle sequence, since the short sequences on the sides are not supposed to be part of the final construct. You should now see something similar to Fig 12.

You have now simulated a cloning experiment that resulted in a recombinant plasmid 🙂.

Question 3:

The first five characters of the cSEGUID of the sequence are “yo_mq”, what are the last five?

What is the size of the resulting recombinant plasmid? Tip! The correct size of this plasmid is the same as the correct sizes for all other examples in this document, except Question 9.

How many SmaI sites can you find in the recombinant plasmid?

In the previous example, the two molecules were combined in this way (Fig 13). The black part of the sequence represents the pUC19 vector.

*

But there is one more way that the molecules can combine (Fig 14). The insert fragment (blue and green) has been cloned inverted compared to the previous example.

*

This will happen for 50% of the recombinant molecules in a real experiment. The lower green strand in Fig 13 is now on top in Fig 14. Please take your time to study Fig 13 and Fig 14 so that you are sure you understand the difference.

Question 4: Simulate the cloning of the molecule in reverse (Fig 14) in pUC19. Tip! First open a fresh copy of the pUC19 plasmid sequence.

What is the size of the resulting recombinant plasmid?

How many SmaI sites can you find in the recombinant plasmid?

The first five characters of the cSEGUID are “_PTxm”, what are the last five?

Some other enzymes such as BamHI or EcoRI cuts the DNA in a staggered manner so sticky ends are produced. BamHI (G^GATCC) cuts DNA like in Fig 15.

The sequence in Fig 16 is the same sequence as in the SmaI example (Fig 4), but with BamH sites instead of SmaI sites at each end:

* It will be digested by BamHI to produce three short DNA molecules (Fig 17):

*

Reopen a fresh copy of the pUC19 sequence file. We will now recreate the same cloning experiment as in the SmaI example, but with BamHI. We have located the BamHI site in Fig 18.

Copy the 15 bp blue sequence in the middle between the BamHI cut sites (Fig 17) and paste the sequence at the cut site for BamHI in pUC19 (Fig 19).

Question 5: Create the sequence of the recombinant molecule in ApE as described above. The first five characters of the resulting sequence cSEGUID are “HAPCm”, what are the last five?

What is the size of the resulting recombinant plasmid?

How many BamHI sites can you find in the recombinant plasmid?

Question 6:

The fragment can also enter in two directions as in the previous example (Fig 13 and Fig 14). Simulate the cloning of the BamHI fragment in reverse. The resulting vector has a cSEGUID where the first five characters are “xLDpt”, what are the last five?

The enzyme KpnI (GGTAC^C) digests DNA like in Fig 20.

The same sequence as in the previous example (Fig 16) but with flanking KpnI sites is shown in Fig 21.

The fragment in Fig 21 is cut like this by KpnI (Fig 22):

Question 7:

Open a new copy of the pUC19 sequence file. Create the recombinant molecule resulting from cloning the fragment in Fig 22 into the KpnI site of pUC19 in the direction indicated (a) and the reverse (b).

Sequences	cSEGUID first five characters	cSEGUID last five characters
pUC19-KpnI(a)	FGhL9	?
pUC19-KpnI(b)	s4jFF	?

Not all restriction enzymes cut inside the recognition sequence. The restriction enzyme AarI cuts for example on the side, four nucleotides away from the recognition sequence (Fig 23). More details about this enzyme can be found at rebase.

AarI does not come in the standard enzyme list of ApE, you have to add a file called “Absolutely_all_Enzymes.txt” to ApE. This file should be present in the same folder as this document. Save it on you desktop or wherever you saved the ApE program. Open ApE and select Enzyme Selection... and then File and open new enzymes file (Fig 24). Locate the “Absolutely_all_Enzymes.txt” and open it. You will see that there are now many more enzymes available.

Reopen the pUC19 sequence file in a new window. Enter the sequence below in a new ApE window:

The sequence has one AarI site and one BglII site, Highlight both sites in the Enzyme selector (Fig26).

Question 8:

Your task is to simulate the cloning of the sequence in Fig 25 into pUC19 using the BamHI site in pUC19. Selection of the correct fragment is trickier than before. Use what you know about the AarI site (Fig 23) to make the correct selection. Simulate cloning in both directions as before (a) and (b).

Sequences	cSEGUID first five characters	cSEGUID last five characters
pUC19-AarI-BglII(a)	VketZ	?
pUC19-BglII-AarI(b)	aSX2K	?

How many BamHI sites are in the final molecule?

In question 8, you ligated a BglII site in the insert DNA fragment to a BamHI site in the pUC19 vector. How come we can do this ? Although the recognition sites are different, they produce the same cohesive ends. We can also say that the two enzymes produce compatible overhangs (Fig 27).

Note that the hybrid site is not recognized by either enzyme. This means we can not cut it with either BglII or BamHI.

In the case of the AarI site in question 8, the overhang sequence is NOT defined by the recognition sequence.

In our case, the AarI site is only compatible with the BamHI and the BglII sites because of the specific sequence of the DNA fragment in the region 4-8 nucleotides to the right of the recognition sequence of AarI (Fig 28).

Question 9:

Your task is to simulate the cloning of the 1.5 kb BglII – EcoRV fragment from the vector pUG6 into the BamHI – SmaI sites of pUC19.

There should be a file called: “vector_pUG6_in_raw_sequence_format_cSEGUID_mCu72Qz7jeW7nIZgnuTO8YgAygM.txt” that contains the sequence for the pUG6. It is also available from GenBank under the accession number AF298793.

This is a directional cloning since the enzymes are not all compatible, see details for how they cut using the same technique as in Question 1. This also means that there is only one result and not two as in the previous examples.

The first five character of the cSEGUID checksum of the resulting sequence is nTgoQ. What are the last five characters? The size of the recombinant vector should be exactly 4184 bp.

Question 10:

This is an individual exercise for each student. The input data can be found in a Google spreadsheet where you can find your name in the leftmost column.

One column called sequenceZ contains a DNA sequence that represents a double stranded linear DNA molecule. The columns re1 and re2 contain two restriction enzymes that cut the sequenceZ. The restriction enzymes also cut the plasmid pUCmu.** The sequence of the pUCmu can be found in the file pUCmu_1669_bp_cSEGUID_wonHpAgf0BcUTNAorUx1-Nkr7xs.txt in the folder of this file. Your task is to simulate the cloning of sequenceZ in pUCmu after digestion of both with the two suggested enzymes (Fig 30). The resulting sequence where sequenceZ is cloned into pUCmu is called pUCmu_Z. Put your sequence in the indicated cell. Please answer with raw DNA sequences as indicated for the first example student "Max Maximus”. If your name is *not* in the list, please inform your instructor.

Follow this link to the Google Spreadsheet. © Björn Johansson 2022