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Homework Assignment #1 and Vector NTI Session for Experiment 1, “Simple Gene Knockouts”

You will be performing a gene knock-out (gene deletion) by transformation and homologous recombination using Aspergillus nidulans. You will be deleting a nonessential gene called wA for “white”; wA mutants produce non-pigmented (white) colonies instead of pigmented (green) colonies. This experiment will allow you to easily detect the mutants you have created by simply observing colony color.

Outline:

Step 1) create a gene-knockout construct using PCR

a) Design and create a PCR product containing DNA 5’ to the gene (5’ flank)

b) Design and create a PCR product containing DNA 3’ to the gene (3’ flank)

c) Fuse the 5’ flank, a selectable marker, and the 3’ flank together by PCR to create a “knock-out construct”

Step 2) transform the construct into A. nidulans cells and plate cells on selective medium

Step 3) identify transformants that are likely to be deletion mutants based on their phenotype

Step 4) isolate DNA from the selected transformants and use PCR to determine whether they contain the deletion mutation

We will use Vector NTI to design this experiment and we will carry out this experiment at the bench. The remainder of this document describes how to use of Vector NTI to design and predict the results of steps 1 and 4 above. Your first homework assignment will require you to perform these Vector NTI operations on a different gene.

Step 1 Overview: Creating gene knockout constructs using fusion PCR. You’ll construct this DNA molecule by “fusion PCR” using the strategy diagramed in the figure. The rationale for using this strategy is to rapidly generate constructs suitable for gene deletions without the need for cloning. This molecule is composed of the sequence that flanks the gene on the 5’ side, a selectable marker for the transformation, and the sequence that flanks the gene on the 3’ side. This approach has been used primarily in yeast but has recently been adapted for filamentous fungi (Yang et al., 2004) and could, in principle, be used to generate constructs useful for gene knockouts in animals. To create this construct, you generate three PCR products: a 1 kb product corresponding to the genomic sequence 5’ to the gene (“5’ flank”), a 1 kb product corresponding to the genomic sequence 3’ to the gene (“3’ flank”), and a PCR product containing the selectable marker. The 5’ flank will be generated using primers P1 and P3 and the 3’ flank will be made using P4 and P6 (see figure). Note that 5’ ends of P3 and P4 contain sequences that are identical to either end of the selectable maker, which makes the one end of each “flanking” PCR product identical to the ends of the selectable marker. This overlap allows all three fragments to be combined in a “fusion PCR reaction” in which all three fragments are added as template and are amplified using primers P2 and P5 (see figure). The fusion PCR reaction results in production of a ~ 4 kb product that you will use as your gene knock-out construct. The details below walk you through this procedure using the gene UBC11 as an example. UBC11 is simply an example gene and is otherwise not relevant to this course.

Step 1a) Design the 5’ flank PCR product and PCR primers P1 and P3. Open AN5495 UBC11, which is a Vector NTI molecule representing 12 kb of the A. nidulans genome that includes UBC11. Select the UBC11 CDS using the graphics pane (click on the arrow that represents UBC11), which will select from 6504 to 7169. We’ll design our primers to amplify 1000 bp 5’ to this, from 5404 to 6404. Click the window showing the selected region and change it to 5404 to 6404. Pull down the analysis menu, choose primer design, choose amplify selection. A box will open showing information in the “primer tab” that allows you to input required parameters. The region selected should show up in the “amplicon must include region of the molecule” boxes. To the left of that is a box that allows you to input how many additional nucleotides to the left of the selection can be searched to find good PCR primers. The box to the right indicates the same for searching to the right of the selection. Type 100 into each of these boxes. The number of output desired should be three. The bottom has to do with specifying the physical and thermodynamic properties of the primers and therefore requires information on the conditions of the PCR reactions. The salt concentration should be 50 mM and the dG temperature should be 25 degrees. The T_M should be between 55 and 65 degrees. The %GC should be between 40 and 60. The length should be between 18 and 23. The primers should be DNA. Click OK, and the process will run, resulting in a new folder, PCR analysis, in the text pane.

There will be three PCR products listed in the text pane, each with a size and rating (171 is the best rating Vector typically gives a PCR strategy) and a set of PCR primers. Right click on product 1 and choose “find PCR product”. This amplicon is now highlighted in the graphics and sequence panes. It should start at 5358 and end at 6404 (but it’s OK if your results are slightly different). The primers are each 21 nucleotides long with a Tm close to 55 degrees. Save this PCR product as UBC11 5’ flankXX (where XX are your initials) by right clicking the product line and choosing “save as molecule in database and open new window”. A box will appear for you to name the product, name it “UBC11 5’ flankXX ”, save it in the BIO 510 2009 subset, and click OK. The new molecule file will open. The primers are features that we will save as oligonucleotides in our database. Do this by selecting the sense primer using the sequence pane, then click the Add to Oligo List icon (or list > add to oligo list), name the primer UBC11 P1XX, click the oligo tab to make sure the sequence in this file is the 21 nt sequence corresponding to top strand at the left end of the molecule, click OK. Repeat this process for the antisense primer, except this time name it P3, and make sure the sequence corresponds to the bottom strand at the right end of the molecule. When you check the sequence in the oligo tab, the top strand should be showing and you’ll have to click the “Reverse Complementary” box before clicking OK.

The P3 oligo you designed lacks the 21 base sequence that is identical to the 5’ end of the selectable maker you will use. This corresponds to the red part of the P3 primer shown in the figure above and it is important for the fusion PCR reaction that combines the 5’ flanking DNA with the selectable marker. To add this sequence to the 5’ end of your P3 primer, we will select and copy the sequence from the selectable marker sequence and then past that sequence at the 5’ end of the P3 oligo. First, open the selectable marker sequence, called “Af pyrG only”. In the sequence pane, select the first 21 nucleotides and then click the “add to oligo list” button. Name the oligo “5’ end of P3 for Af pyrG”. Click the oligo tab and choose “reverse complement” then click OK. This oligo can be saved and used to add to the 5’ end of any P3 primer you design for doing knockouts using Af pyrG as the selectable marker. To add this sequence to the P3 you just designed, select the “5’ end of P3 for Af pyrG” oligo file, choose “edit” from the menu above the sequence list, click the oligo tab, select the sequence, copy it, and then close sequence. Select the P3 oligo you just designed, and click on “edit” and then paste the sequence you copied in front of (at the 5’ end of) your P3 sequence. Click OK, and notice that your P3 primer is now ~42 bases long.

To save the P1 and modified P3 oligos you have designed to the database, select the P1 oligo, click save, choose the BIO 510 2009 subset, and click OK. Do the same for P3. Now you are ready to design the 3’ flanking PCR product.

Step 1b) Design the 3’ flank PCR product and PCR primers P4 and P6. Designing primers for the 3’ flanking fragment follows a similar procedure, but let’s go through it for practice (you’ll be doing this exercise on another gene for homework, so take advantage of the chance to practice). Select the UBC11 CDS, change the selection to 1 kb on the right (7269 – 8269) using the selection window as above, pull down the analysis menu > primer design > amplify selection. Make sure parameters are as they were, click OK. The first product is 1032 bp in length (from 7252 to 8283) and the primers have Tm’s at 55 degrees (but it’s OK if your results are slightly different). Right click the product, save to database and open window, name it UBC11 3’ flankXX, click OK, put it in the BIO 510 2009 subset, click OK. Put the primers in the oligo list by clicking on them, clicking on the add to oligo list icon, name them. The sense primer should be UBC11 P4XX, and the antisense primer should be UBC11 P6XX. Click the the oligo tab, make sure the sequence shown is the correct strand (top strand for P4, reverse complementary strand for P6), click OK.

As with the P3 primer, the P4 primer is not complete. It lacks the 21 base sequence that is identical to the 3’ end of the selectable maker that you will use. This corresponds to the red part of the P4 primer shown in the figure above and it is important for the fusion PCR reaction that combines the 3’ flanking DNA with the selectable marker. To add this sequence to the 5’ end of your P3 primer, we will select and copy the 21 nucleotide sequence from the very 3’ end of the Af pyrG selectable marker then past that sequence at the 5’ end of the P4 oligo. Bring up the “Af pyrG” sequence (should still be open), select the last 21 nucleotides, click the “add to oligo list” button. Name the oligo “5’ end of P4 for Af pyrG”. Click the oligo tab and check that the sequence corresponds to the top strand sequence, and click OK. This oligo can be saved and used to add to the 5’ end of any P4 primer you design for doing knockouts using Af pyrG as the selectable marker. To add this sequence to the P4 you just designed, select the “5’ end of P4 for Af pyrG” oligo file, choose “edit” from the menu above the sequence list, click the oligo tab, select the sequence, copy it, and then close sequence. Select the P4 oligo you just designed, and click on “edit” and then paste the sequence you copied in front of (at the 5’ end of) your P4 sequence. Click OK, and notice that your P4 primer is now ~42 bases long.

To save these oligos to the database, select the P4 oligo, click save, choose the BIO 510 2009 subset, and click OK. Do the same for P6.

Note on modifying PCR primers by adding sequences to the 5’ ends. Primers P3 and P4 contain sequences at their 5’ ends that are homologous to the Af pyrG selectable marker. These sequences are important for creating 5’ flank and 3’ flank molecules that have homology to the selectable marker, which is required for the fusion PCR reaction to occur. We added the Af pyrG sequences to P3 and P4 by editing the oligonucleotides after designing them and saving them in our oligo list. An alternative method using Vector NTI is to add them to the primers during the design of the 5’ flank and 3’ flank PCR reactions. We will use this method of primer design later in the course, in the section on using site-specific recombinases in genome engineering and subcloning.

Step 1 C) Fuse the 5’ flank, a selectable marker, and the 3’ flank together to make UBC11 P1 to P6 and use this to design primers P2 and P5. There are two primers remaining to be designed; P2 and P5. These are “nested” primers (hybridize just inside of P1 and P6). They will be used in the fusion PCR reaction, in which you will use the 5’ flank, the selectable marker, and the 3’ flank as templates and P2 and P5 as primers. To generate these primers, we first need to make a UBC11 P1 to P6 molecue, which is a combination of the UBC11 5’ flank, the selectable marker (called Af pyrG), and the UBC11 3’ flank. Open all three files, put the 5’ flank sequence into the fragment goal list, put the Af pyrG in the list, and then put the 3’ flank in there. Make sure that all of each molecule (from start to end) is added to the list. Open the fragment goal list, click run, name the file UBC11 P1 to P6XX, CLICK “LINEAR”, then “construct:, save in BIO 510 2009, click OK. This will save the file in the database and open a window showing the file. Close the fragment goal list. Before using this file, make sure it’s correct. First, look at the components in the text pane. Should be 5’ flank, then the Af pyrG, then 3’ flank. Second, check by adding the PCR primers P1, P3, P4 and P6 as motifs using the analysis menu or display set up icon, and make sure they hybridize where they are supposed to hybridize (the correct location and the correct strand). Also make sure they are100% matches to the P1 to P6 molecule. Check that P1 and P4 match the top strand in the right locations and that P3 and P6 match the bottom strands at the right locations. Note that P3 primer should match to the 3’ end of the 5’ flank and the 5’ end of the Af pyrG. Likewise, the P4 primer should match the 3’ end of the Af pyrG and the 5 ‘ end of the 3’ flank.

Now we can design the P2 and P5 primers to amplify this molecule. Select from 101 to to 4004 and use the analysis, primer design, amplify selection feature as you did previously. The first product 3949 bp from 100 to 4048, will work just fine (your product may differ slightly). Save it into the database and name it UBC11 P2 to P5XX. The sense primer is P2 and the antisense primer is P5. Save these by adding them to the oligo list and saving the from the list tot the database. Make sure that the P5 primer is the reverse complement to the initial sequence shown (matches the bottom strand of UBC11 P2 to P5). To check the entire strategy, go back to the UBC11 P1 to P6 file, add P2 and P5 to the motifs, and make sure they match at the right position and to the correct strand. Now you have designed complete set of molecules and oligonucleotides to perform a gene knock-out of UBC11.

Overview of Step 2: Transformation of deletion construct into A. nidulans. The deletion construct will be transformed into A. nidulans cells and the cells will be plated on selective medium, where only cells that take up and express the selectable marker will be able to grow. Since the gene knock out construct is not an autonomously replicating molecule, it must integrate into a chromosome in order to be maintained by the transformant. Only cells that contain integrated copies of the construct will grow into visible colonies. Integration may occur by non-homologous recombination, which will not result in a knock out of the target gene. Integration may also occur by homologous recombination, which results in replacement of the target gene with the selectable marker, as shown in the figure. Thus, integration by homologous recombination results in deletion of the target gene and an insertion of the selectable marker in its place.

Overview of Step 4: Confirming that mutants contain the deletion mutation. You will knocking-out the wA gene and we will use the known phenotype of wA gene knockout mutants do identify transformants that carry the deletions. However, it is important for you to consider how you would confirm that the transformants contain the engineered mutation. There are two assays used to confirm that you have the proper deletion mutant. The correct results for both assays is required in order for you to be able to interpret the phenotype of your mutants as being due to the deletion you created First, PCR is done using genomic DNA from your strains to amplify the genetic locus to determine whether the gene has been replaced by the deletion construct (see figure). We will design this PCR reaction and predict the results for a deletion mutant and a wild type strain using Vector NTI and our example gene, UBC11. Second, you would perform a southern blot using the selectable marker (Af pyrG in this case) as a probe to determine whether the deletion construct integrated into other sites in addition to the site you desired. These events are referred to as ectopic (other site) integrations, and are common occurrences during transformation of most eukaryotic cells. We will not be performing the Southern blot in the lab. .

Step 4: Using Vector NTI to design and predict the results of PCR reactions that can be used to determine whether transformants contain the knock-out mutation. The most straight forward PCR reaction to perform is one using the P1 and P6 primers. Remember that the knock-out construct does not contain sequences that correspond to P1 and P6, so the only site that these primers can hybridize is the UBC11 locus. This will be true regardless of whether the strain being analyzed contains the deletion. The size of the PCR product produced by this reaction will be 2926 bp if the strain has a wild type UBC11 locus, whereas it will be 4174 bp if it contains the deletion. Do determine this using Vector NTI, open the AN5495 UBC11 region file and the UBC11 P1 to P6 file. Add UBC11 P1 and P6 as motifs and determine the distance from the start of P1 to the end of P6 in both molecules. You can also generate a PCR product that corresponds to P1 to P6 amplification of the wild type locus by selecting the sequence between P1 and P6 and using the Primer Design menu to amplify the selected sequence using “user defined” primers. To do this, select the sequence to be amplified, make sure that you allow Vector to search 30 base pairs (at least the size of the primers) before and after the selection, and add the UBC11 P1 and UBC11 P6 primers by clicking on the box and finding them in your oligonucleotide database.

HOMEWORK 1: Due via email to me at pmmira00@uky.edu by the start of class Friday, October 29^th

Part (a) Design PCR primers to create a deletion construct of the A. nidulans gene, AN3753 trtA. This is the gene encoding telomerase reverse transcriptase, which you will be working on later in this course. You have Vector NTI file of the Aspergillus genomic region containing AN3753 gene and the Af pyrG in your database. Save the 5’ flank, 3’ flank, P1 to P6, and P2 to P5 DNA molecules. Name the molecules and the oligonucleotides as in example above except they should be called trtA instead of UBC11. For example, the 5’flank of trtA should be “trtA 5’ flankXX”, where XX are your initials. The P1 primer will be trtA P1XX.

Part (b) Predict the results of P1 to P6 PCR reaction using the genome of a wild type strains and a strain in which you have deleted trtA using your design from part (a). Include the sizes of the products in the deletion mutant and the wild type strain in the text of your email to me.

Submitting your homework Save all the molecules and primers in subsets called “Homework 1”. Create archive files containing all the molecules and primers you created (one archive file for molecules, one archive file for primers). Email the archive files to me at pmmira00@uky.edu by the start of class on Friday, October 29^th.

PLEASE NOTE: You should send me only two vector NTI files, one archive of the molecules you created and one archive of the oligos. Sending more Vector NTI files than this will result in a 50% reduction in your score on the homework assignment.