Vector NTI and
Homework Assignments
Purpose: To introduce
you to Vector NTI software, which is common application for storing, manipulating,
and analyzing DNA/RNA/PROTEIN sequences.
We will be using Vector NTI thoughout the second half of BIO 510 to
visualize the DNA molecules we will be working with and to predict and analyze
the results of our manipulations of those DNA molecules.
Introduction to Vector NTI. Start vector NTI using your desktop shortcut. Open the database by clicking the toolbar icon. The window on the left lists the types of databases and the subsets in each database. The DNA/RNA database is selected now. Pull down the window and notice that there are databases for proteins, enzymes, oligonucleotides, gel Markers, etc. The window on the right lists the individual items in each database. Choose DNA/RNA molecules. The window on the right lists ALL DNA/RNA molecules loaded in this database, which includes the files in each subset. Choose BIO 510 2007 subset. Let’s look at the plasmid that will be the focus of the first three experiments, pNIG6. Double click on it.
The pNIG6 window has three panes: the text pane, the graphics pane, and the sequence pane. Make it so that you can see all three. Move between panes by clicking in the pane or on the pane icons. Note that the icons above the text and graphics panes change as you move from text to graphics to sequence. This reflects the fact that different function can be performed depending on which pane is active. The content of pull down menus can also change depending on which pane is active.
Click on the display setup icon next to the seq pane. Pull down the setup profile window and choose BIO 510 2007. This sets up the display options, for example, how the sequence is viewed, what restriction sites will be shown, what motifs are shown, and so on. This profile does not include any motifs (short DNA or Protein sequences), we’ll add them together. Click OK to get pNIG6 to be displayed the way we want to see all our molecules displayed.
Open the Restriction/Methylation Map folder in the text pane to see a list of all the enzymes being displayed, the number of times they cut, and their recognition sequence. Open the EcoRI folder to see the position of the two EcoRI sites. Right click on the sit at 4637, choose find REN…. Now the EcoRI site at 4637 is highlighted on the graphics display AND in the sequence pane. Lets add the BamHI restriction enzyme to the BIO 510 2007 profile. Click the display setup icon, find BIO 510 2007 again, click RMap setup, click Add, scroll to BamHI, click it, click OK, OK again. Click save settings as BIO 510 2007 OK, click yes to replace the old settings with the new ones, click the top of the display setup box to make it active and then OK. The text, graphics, and sequence panes all have BamHI information displayed now.
You can also adjust the display by using the Analysis pull down mendu. Lets use the menu to remove BamHI from the current display. Click Analysis menu, restriction analysis, restriction sites, BamHI, remove and then OK. Note that BamHI is still in the BIO 510 2007 profile. Open the display set up, choose the profile, click rmap set up. Get rid of BamHI (we don’t need it). OK. Save settings as BIO 510 2007, OK, yes, click the display box, OK. Make sure BamHI is gone by looking in the Restriction /Methylation Map folder.
Open the Feature Map folder and you’ll see the folders it contains, each of which has information of part of the pNIG6 plasmid. pNIG6 is a plasmid that has a selectable marker for transforming Aspergillus nidulans (a fungus we’ll work with this semester), and the gene encoding the Aspergillus cdc2 gene, called nimX. Open the miscellaneous feature folder, which contains selectable marker gene, Ncrassa pyr4, which is from the fungus, N. crassa. Ncrassa pyr4 complements the uracil auxotrophy due to mutations in A. nidulans pyrG (encode the same enzymatic activity). Therefore, pNIG6 can be used to transform a pyrG mutant of A. nidulans by selecting for growth on medium lacking uracil. Right click Ncrassa pyr4 and choose Find Ncrassa pyr4… and the gene will be highlighted on the graphics and seq panes. Click anywhere in the sequence pane and the selection is removed. Note that a cursor is in the sequence pane, indicating which nucleotide of pNIG6 it’s next to. That nucleotide number appears in the box on the lower right of your screen. Now click on the green arrow in the graphics pane that corresponds to Ncrassa pyr4 and you’ll again highlight this region of pNIG6 in both the graphics and sequence panes. Any feature that shows up on a DNA/RNA/Protein molecule in Vector NTI can be highlighted in these ways. Take a moment to find and highlight the other features of pNIG6 (mRNA and exons of nimX).
You can select restriction sites and features in the Graphics pane (we made pNIG6 that way) and in the Sequence pane. Select the fist 100 bps in the sequence pane. Notice that they are highlighted in the seq pane and selected on the graphics display. You could combine any selected sequence with sequences to create new recombinant DNA molecules (we’ll do an example of this soon) in vector. Use of Vector NTI to plan and store files of all your clones is useful if your are doing any molecular biology in your research.
You can change the display from circular to linear, which automatically chooses to run from 1 on the left to 7159 (end) on the right. Click the graphics pane icon, and the linear display icon. This can be useful but should not be used unless necessary. Some of our DNA sequences ARE linear (for example the nimX region that we are about to use), and so the linear display makes the most sense for files like that. To determine whether a sequence was created as a circle or a linear molecule, open the General Description folder in the text pane and look at the last entry. For pNIG6, this should be circular. Make sure the display is circular before continuing.
Making recombinant DNA molecules. Let’s re-make pNIG6 by cloning the nimX gene as a PstI to XbaI fragment from the nimX region into pRG3 cut with PstI and XbaI. You can see that pNIG6 is composed of these DNAs by clicking on the component fragments folder in the text pane. It lists both components, a fragment (part of) pRG3 and a fragment of nimX region. pRG3 and nimX region are both Vector NTI DNA molecules. The names should also be hyperlinks to open those files and you can open them using those links. Do so. pRG3 is an E. coli/A. nidulans shuttle vector and is the vector used to clone the insert, which is an A. nidulans genomic DNA fragment containing the nimX gene. It confers resistance to ampicillin in E. coli and uracil prototrophy to pyrG mutants of A. nidulans. nimX region was taken from the A. nidulans genome sequence and includes the entire nimX gene plus flanking DNA. pNIG6 includes pRG3 from pRG3 position 45 to 28 (always clockwise) and nimX region from 2726 to 5221. The position of each of these components in pNIG6 is also indicated. If the files, nimX region and pRG3 are not already open, go to the exploring local vector database window and open them so we can re-create pNIG6.
First, get the PstI to XbaI fragment of pRG3. In the graphics pane of pRG3, click on PstI
site, hold down the shift button, and click on the XbaI site. The plasmid from PstI to XbaI will be
highlighted. We’ll drop this fragment
into the Gragment Goal List and then come back and get the nimX gene fragment. To do
that, click the Add Fragment to Goal List icon above the graphics pane, make
sure that the
Use the window menu to bring up the nimX region display. Change the display setting to BIO 510 2007 if they are not already set to them. Let’s get the XbaI to PstI fragment into the goal list. Click XbaI site at 2726, hold shift, click the PstI site at 5222 – the fragment is highlighted. Note the display at the bottom right that reads 2726 bp – 5221 bp (2496 bp). This is the position of the fragment and its size. Add this to the goal list as you did for the pGR3 fragment.
Open goal list using the icon. The pRG3 vector runs from PstI to XbaI and the nimX insert runs from XbaI to PstI, which should clone just right (PstI-Xba ligates to XbaI-PstI and then PstI ligates to PstI to close the circle). Click Run to construct the new plasmid, name it pNIG6XX (XX = your initials), make sure it is listed as a circular molecule, and the click construct. You will be asked where you want to put this sequence. Type BIO 510 2007 XX (XX = your initials) and click OK. The new version of pNIG6 will be in your subset as well as in the overall database. Each student should make a pNIG6XX molecule and save it in a new subset created as indiated above. Set the display of your new pNIG6 molecules to BIO 510 2007 so you can compare the new to the old.
You can save your pNIG6 molecule to a separate file that can be shared between Vector NTI users by saving it as a “archive” file. Whenever you turn in Vector NTI molecule files to me as part of homework or exam 2, you’ll save the files as archives and give me the archive file. Lets create an archive file containing your pNIG6. Bring the database explorer window up and click on the DNA/RNA molecule subset BIO 510 2007, where you just saved your pNIG6 molecule. Click on your pNIG6XX molecule, then pull down the DNA/RNA menu, choose Export, then Selection into archive, name it pNIG6XX, and save it in the My Documents folder. It will automatically save the pNIG6XX and all the other Vector Molecules that were used to create it (pRG3 and nimX region, in this case). To open an archive file, just double click the file and Vector NTI will automatically open and walk you through importing the molecules from the archive into the desired database and subset.
Predicting the results of Restriction Digests You can use Vector NTI to predict the results of restriction digests. Lets do this for pNIG6. Open pNIG6, pull down the analysis menu, select restriction analysis, select restriction fragments, unselect all, click on XbaI and PstI to select those two enzymes, click OK. The results appear in the text pane, in a folder called restriction fragments. You should see two fragments listed, one 4663 bp in length from PstI at position 1 to XbaI at position 4664. The other is 2496 bp in length, from XbaI at position 4664 to the PstI site at position 1. To printout the result of your restriction digests, make the text pane active, click the print preview icon, and print that page.
Experiment 1: Site-directed Mutagenesis
Purpose: To create a mutant version of pNIG6 that contains a missense mutation (tyrosine converted to histidine) at position 306 in the nimX amino acid sequence. To do this, we will be doing oligonucleotide directed (site directed) mutagenesis, where mutant PCR primers will be used to prime DNA synthesis by PCR We’ll test whether the mutation inactivates the nimX gene by determining whether the mutant nimX gene we created can complement a known nimX mutation in Aspergillus cells.
Let’s look at how we’ll use pNIG6 in our first experiment, site-directed mutagenesis. In this experiment, we are going to introduce a mutation in the nimX gene, which is the gene you just cloned in silico using Vector NTI into the vector pRG3 to make pNIG6. Based on sequence comparisons, nimX is the A. nidulans ortholog of cdc2, the cyclin-dependent kinase first discovered in S. pombe. To test the hypothesis that nimX is orthologous to cdc2, we’ll introduce a mutation in nimX that is the same mutation as one that is known to inactivate the yeast cdc2. We’ll test whether the mutation inactivates nimX by transforming the mutant gene into an A. nidulans strain that carries a mutation in nimX. If the nimX mutant allele we create cannot complement the known nimX mutation in A. nidulans, then the mutation we created inactivates nimX. If the mutation known to inactivate yeast cdc2 also inactivates nimX, this would support the hypothesis that nimX is a cdc2 ortholog (Osmani et al. (1994) J. Cell Science 107:1519-1528).
One S. pombe cdc2 mutation known to inactivate cdc2 is a substitution of histidine for the tyrosine at position 306 of the polypeptide (it actually creates a temperature sensitive mutation, but that is not important for now). The shorthand for this amino acid substitution is Y306H. A mutation that would cause this substitution is a TA to CG transition at nucleotide position 6232 in the pNIG6 plasmid. Find the TA base pair at 6232 using the sequence pane. Highlight that base pair.
In order to see how this TA to CG transition would cause a Y to H substitution, we need to highlight the correct the open reading frame in this exon and translate it so that both the DNA and amino acid sequence will be displayed in the sequence pain. To do this, highlight exon 4 by clicking on it in the graphics pane. The second base highlighted, the A at 6031, is the first base in the first codon in the correct reading frame in exon 4. This is the +1 frame for this sequence (+1 frame starts at nucleotide one of the plasmid sequence, +1 starts at nucleotide 2, etc). To translate this segment correctly, pull down the analysis menu, choose translation, in sequence pane, 6 frame translations, unhighlight all but frame +1, click translate. The highlighted exon 4 should now show the amino acid above each codon in the +1 frame.
The display is probably the three letter code, which should read Ile, Leu Gly Thr Pro… at the start. Let’s change the display to the single letter code (what the Pro’s use). Click the display setup icon, choose BIO 510 2007 profile, click sequence setup, click 1 letter aa code under translation, click OK, save settings as, yes, OK, click the display set up box, OK. Note that translation of every codon in the +1 frame is shown above the sequence, even though there is an in frame stop codon that ends the nimX ORF at position 6289 (the start of TAA termination codon).
If you wanted to just show the translation of the nimX codons in exon four, you would have to highlight the sequence from position 6031 to 6291. Let’s do that. First, clear the translation by pulling down the view menu and clicking clear all translations. If the view menu does not list clear all translations, then you are not actively working in the sequence pane. Click on the sequence pane icon, then pull down view, then clear all translations. Now, click the box at the lower right of the vector window that shows the sequence that is currently highlighted, 6030 – 6564 (535 bp), which corresponds to all of exon 4. That opens a box that allows you to type in the nucleotide position number you want selected, which we just said was 6031 to 6291. Do that and click OK. Now pull down analyses, translation, in sequence pain, direct strand. This translates the selected sequence, using the first base selected as position 1 as the first codon translated. The translation should start ILGTP and finish GFH* (* means stop).
Highlight the TA base pair at 6232 again. Note that the T is the first base in the TAC tyrosine codon. Changing the T to a C creates a CAC histidine codon (a table of the standard genetic code is on the last page of this handout). We will make a TA to CG transition by using mutagenic oligonucleotides to prime synthesis of pNIG6 in a PCR reaction. The oligonucleotides will match the sequence in this region but will incorporate a CG base pair at this position instead of TA. The Stratagene QuickChange manual explains how oligonucleotides are used to accomplish this and we’ll discuss this in lab. There are four different mutagenic oligonucleotides in module 1, Y306H NsiI, Y306H NsiI Comp, and Y306H SacI and Y306H SacI Comp; we’ll look at the Y306H NsiI oligos now. You’ll perform similar analysis with Y3006H SacI oligos and design your own mutagenic oligonucleotides for homework (see below). Y306H NsiI (and the complementary oligo, Y306H NsiI Comp) match the region around 6232 in pNIG6. We’ll add these oligonucleotides to our display, inserting them as “motifs”. Vector will compare these oligo seqs and show the best match in pNIG6.
Pull down analysis, choose motifs, choose oligo database, find the BIO 510 2007 subset, click on Y306H NsiI and Y306H NsiI comp and click OK and OK again. These oligonucleotides have been added to all three panes, the text pane in a Motifs folder, the graphics pane in a manner similar to restriction sites, and in the sequence pane, where they are represented by a line showing the sequence that they match best with the oligo name above (matches top strand) or below (matches bottom strand) the line. The text and sequence panes also indicate the percentage identity between the oligo motif and the sequence it matches. Click on the Y306H NsiI site in the graphics pane and the sequence pane will show the position of the best match for both oligos.
A problem with the motif display feature of Vector NTI is that it does not show the position of the mismatches between Y306H NsiI and the pNIG6 sequence. To see this, we’ll align the oligonucleotides and pNIG6 sequences using the align function. The align function can align DNA sequence files, but not an oligonucleotide files, so first we’ll have to create a DNA sequence file containing the Y306H NsiI oligo sequence. First, copy the oligo sequence by clicking on exploring the database, choose oligos, BIO 510 2007 subset, and then double click Y306H NsiI. This opens a box with the oligo sequence already highlighted. Press control C on the key pad to copy it, close the box, click on the vector NTI window to make it active, pull down the file menu, create new sequence, using sequence editor, type in Y306H NsiI as a name, click the DNA/RNA molecule tab, click linear, click the sequence and maps tab, click Edit Sequence, click paste, OK, OK, yes. This opens the sequence file and saves the file in the main DNA/RNA molecules database. To put the sequence in the BIO 510 2007 subset, ckick on exploring vector database, choose the DNA/RNA molecules database, find Y306H NsiI in the sequence list on the right and drag it into the BIO 510 2007 subset. Now you have that sequence and pNIG6 to align using the align function.
To align the sequences, pull down the align menu, click on open new alignment window. You need to drag pNIG6 and Y306H NsiI into the upper left hand panel of the alignment window. To do this, click on the exploring local vector database icon in the taskbar or the database icon in Vector NTI window to bring the database up. Find and drag pNIG6 into the upper left pane of the alignment window. It should be listed there after you drag and drop it. Do the same for Y306H NsiI sequence. Click on the alignment window to make it active. Highlight both sequences. In the alignment window, pull down the align menu and choose align selected sequences and wait for the alignment to be completed. There will be a graphical display at the upper right and a sequence alignment at the bottom. In the graphical display, there should be a colored bar showing the region where pNIG6 and Y306H sequence align. Click on the colored bar and it will take you to that region in the sequence pane. You should see pNIG6 sequence and Y306H NsiI sequence, with the matches highlighted. Save this alignment and name it Y306H NsiI group # so that you can open this “alignment project” later. You can also open and save this alignment file to removable disks, which you will have to do to complete your homework assignment.
The alignment shows that two bases are not identical, the other 35 are identical. One mismatch is at 6232 (point at the unhighlited T on the right and a box will display the position number), where there is a T in the pNIG6 sequence but a C in the Y306H sequence. This is the mutation we wish to make. The other mismatch is at 6228 and is also a T to C transition. This mutation is silent with respect to the coding sequence (we’ll see that in a minute) but it removes a NsiI site from pNIG6. Because the mutagenic oligonucleotide becomes incorporated into the plasmids created by PCR in our QuickChange mutagenesis experiment, both T to C transitions are incorporated together. The T to C at 6232 is only detectable by sequencing, whereas the loss of the NsiI site is detectable by a simple restriction digest. So, we screen clones created using the Y306H NsiI oligonucleotides by cutting with NsiI and, any clone that is missing this NsiI site is safely assumed to also carry the T to C at 6232 (this can be confirmed by sequencing this region of the plasmid).
Let’s make a mutant version of pNIG6 that would be created by using the Y306H NsiI oligonucleotides and then translate exon 4 to see the result of the two mutations. Close the alignment window. Click the Vector NTI window to make it active. Save the pNIG6 as pNIG6 Y306H NsiI group#. If 6232 is not currently in view in the sequence pain, click on the Y306H NsiI motif in the graphics pane or click the box at the bottom right of the vector window that indicates the position of the cursor and type in 6232 and hit enter. Highlight the TA base pair by using the cursor and holding down the left mouse button. Pull down the edit menu, choose New, then Replace sequence. Say yes to the warning of disconnection from the parents, delete the T and type in a C and click OK. Say Keep All to the options of keeping the exon 4 and mRNA features intact. The TA at 6232 should now be gone and replaced by CG. Do the same for the TA base pair at 6228. Notice that the NsiI site is gone in the sequence, graphics, and text panes. Also notice that the mutagenic oligonucleotides match the mutant version of pNIG6 100%. Save the file to the BIO 510 2007 subset by clicking the Save icon, pulling down the DNA/RNA database window and selecting the BIO 510 2007 subset, changing the name of pNIG6 to pNIG6 Y306H NsiI (group#). You’ll be saving a similar file in this manner as part of your first homework assignment.
Lets check the translation of this region to make sure that the Y is replaced by an H and that the TA to CG transition at 6228 is silent. Select exon 4 in the graphics pane, pull down analysis, translation, in sequence pane, six frame, unclick all but the +1 frame, OK. Note that at position 6232, the codon is now CAC which codes for H. At 6228, the T to C transition changed a CAT histidine codon to a CAC histidine codon, which is silent with respect to translation. So, incorporation of Y306H NsiI oligonucleotides into pNIG6 during the QuickChange reaction will result in two transition mutations, one of which is translationally silent but destroys a NsiI site, other of which causes the desired Y306H change in the coding sequence. Some of you will be doing this mutation in the lab and will screen the potential mutants by cutting with NsiI and looking for a loss of the NsiI site.
Design of mutagenic oligonucleotides. Lets look at how Y306H NsiI and its complement were designed using the Mutageneis function of Vector. Mutagenesis searches open reading frames in selected/highlighted sequences for silent mutations that either create or destroy restrictions sites. The reading frame is specified by the first base of the selected region. To see how this worked for Y306H NsiI, close all sequence files except for the original pNIG6 and make sure that Y306H NsiI and its complement are shown as motifs. Click on exon 4 and change the selected region to start at 6031 using the selection box at the bottom right. Now pull down analysis, choses translation, in sequence pane, direct strand. The correct ORF of exon 4 should start with the sequence ILGTPDE…Now lets run the mutagenesis function. With the same region highlighted, pull down analysis, choose mutagenesis, choose direct strand, select all the enzymes except LoxP, and click OK. A mutagenesis folder appears in the text pane which contains a list of the silent mutations and the restriction sites they cause to either appear or disappear. Scroll down and find the T to C mutation at 6228 that makes a NsiI site disappear. Right click the mutation and choose find, and that site appears in the sequence pane. Because this silent mutation was close to the desired Y306H mutation (6232), I was able to design a mutagenic oligonucleotide that made both mutations and satisfied the specifications dictated in the QuickChange manual. Confirm for yourself that Y306H NsiI meets these criteria when you have time during this lab or at home.
Once a sequence is found that satisfies these criteria, oligonucleotides corresponding to that sequence and its complement were ordered for use in the QuickChange mutagenesis procedures. Let’s select the wild type sequence that corresponds to Y306H NsiI and edit it to create the mutagenic oligonucleotide and then save it as a new name to the oligonucleotide database (you’ll be doing this in your first homework assignment). First, highlight the sequence under the Y306H NsiI oligo in the sequence pane (should include 6213-6249). Click the add oligo to list icon, name it Y306H Nsi your-group-number, click the oligo tab, notice that the sequence is the forward sequence (top strand) – you could change this to the complement by clicking the reverse complement window, BUT DON”T – click OK. A box tells you that your sequence has been added to the oligo list, which is a temporary storage area like the fragment list. Save this oligo to the database: open the oligo list by clicking that icon, click once on the oligo you just entered, click save, choose the BIO 510 2007 subset, click OK.
NOTE: In designing Y306H NsiI, I was able to come up with an oligonucleotide considerably less than 45 nucleotides long, that satisfied QuickChange criteria and contained both the silent mutation (loss of NsiI) and the desired missense mutation (Y306H) approximately in the middle. When you design a mutagenic oligonucleotide using this procedure AS PART OF YOUR FIRST HOMEWORK ASSIGNMENT, make it exactly 45 nucleotides long with the two mutations as close to the middle as possible.
HOMEWORK 1 DUE by the start of class on Monday October 29th . You can email it to me anytime before that at pmmira00@uky.edu or bring the files to class on a flash drive. You can work together with your lab group using the same lap top you use in class or another computer, but the work you turn in must be your own work. If you want to use a 510 lab top, contact the TA to arrange to use it IN ROOM 224. The lap top cannot leave room 224. I suggest that you download your own free version of Vector NTI and put in on your lap top computer or a desk top computer. We’ll cover this process in class.
Part 1) The other mutagenic oligonucleotides to be used in the QuickChange module is Y306H SacI and Y306H SacI Comp. These oligos will also cause the T to C transition at 6232 which causes the Y306H substitution. It also introduces a silent mutation that adds a new SacI site into pNIG6. Use Vector NTI to go through the same steps with Y306H SacI as done with Y306H NsiI in the description above.
To complete the assignment, bring a CD/flash card to class that contains:
a) the alignment file showing the mismatches between Y306H SacI and pNIG6 (save it as Y306H SacI (your name)
b) a mutant version of pNIG6 that has the mutations which would be created using the Y306H SacI oligos (call it pNIG6 SacI your-last-name). The pNIG plasmid should be saved as es pNIG (your initials). Save that plasmid as an archive file of the same name and turn that into me.
Part 2) Create a mutagenic oligonucleotide suitable for use with the QuickChange system to create the mutation F82L. The F codon is at 5450-5452. Your oligonucleotide should cause a T to C transition at 5450 and a second, silent mutation that creates or destroys one the standard restriction sites in the BIO 510 2007 display set up (except for LoxP). The correct reading frame for exon 2 of nimX starts at 5246, and the translation of exon 2 begins with the protein sequence TYGVVYK. NOTE: use the information from the QuickChange manual on design of mutagenic oligonucleotides to guide you in your design, except make the oligo 45 nucleotides long. To complete this assignment, save the oligonucleotide sequence corresponding to the top strand of pNIG6 as F82L (name of restriction site) (your initials) in the BIO 510 2007 oligonucleotide subset (for example F82L BamHI PM).. To save this oligo file to your disk, click on exploring local vector database, choose oligonucleotides, BIO 510 2007 subset, highlight your oligonucleotide, pull down the oligo menu, click export, selection into archive, choose your CD or flash card, name it as above, and save. Save the oligo as an archive file with the same name as the oligo.
Vector NTI Session
for Experiment 2: Random Mutagenesis
Let’s use Vector NTI to briefly look at our GeneMorph experiment, where we will be introducing random mutations into a portion of the nimX gene by error-prone PCR. In this experiment, we will be using primers called domain 5’ and domain 3’ to amplify a “domain” of nimX under conditions that promote mistakes in replication using the GeneMorph kit. Let’s look at what region of nimX will be amplified under these conditions. Open pNIG6. The display shoud be the BIO 510 2007 display: if not, change to that display. Pull down analysis, choose motifs, then oligo database, BIO 510 2007 subset, highlight domain 5’ and domain 3’ oligos, click OK, OK again. The oligos will display in all three panes. The region between the 5’ and 3’ domain oligos will be amplified and mutated in this experiment. Note which regions of nimX are contained in that amplicon and consider how mutation in each of those regions could result in a reduction or complete loss of nimX function.
Let’s create a file containing the sequence to be amplified. Select the entire nimX insert by clicking on the XbaI site (4664) and the PstI site (1). You could also have done this by opening the component fragment folder in the text box, right clicking on the nimX region, and choosing find…. Now pull down the analysis menu, primer design, PCR using existing oligos. Click sense primers, add, domain 5’, OK, OK. Click antisense primers, domain 3’, OK, OK. OK again will run the analysis. The text pane should have a folder under PCR analysis that has a folder with Product #1 of length 988. Right click the text name of that folder and choose find PCR product. The amplicon sequence will now be highlighted in the graphics and sequence panes. To save this amplicon as a molecule, right click the Product #1 name, choose save as molecule in Database and create window. Name it pNIG6 GeneMorph Domain (group #) and click OK, choose the BIO 510 2007 subset, click OK. Notice that the features displayed include only exon 1 and not part exon 2, even though part of exon 2 is included in this PCR product. This is a property of Vector NTI – if only part of a feature is used to make a new molecule, that partial feature is not displayed. You can add that feature back. To do this, click the graphics pane icon to activate the graphics pane, click the add feature icon, choose the exon feature type, name the feature part of exon 2, type 587 in the from box, 988 in the to box, and click OK. You can save this edited version by clicking the save icon, clicking OK, and overwrite.
You know have the PCR product that will be amplified in the GeneMorph experiment as a DNA molecule in your database, which you can use to determine if your PCR reaction in the GeneMorph experiment has produced a band of the correct size.
That’s it for this Vector NTI session.
Vector NTI Session
for Experiment 3: Transposon Mutagenesis
You’ll be using the transposon in pGPS2.1 to identify regions of the insert in pNIG6 that are required for the function of the Aspergillus nidulans nimX gene. Open Vector NTI and open the DNA molecules: pGPS2.1, pNIG6, GPS Hop in pNIG6 insert, and GPS Hop in pNIG6 vector. First look at pGPS2.1. The transposon is the region containing the two direct repeats 7R and 7L and everything in between (from 3093-4467). The sequencing primer binding sites are within these repeats. To see the primer sites, pull down analysis, choose motifs, oligo database, BIO 510 2007 subbase, select Primer S and Primer N, OK, and OK. Select 7R and look in the seq pane, note that Primer N is a 100% match in 7R, and corresponds to the bottom strand, meaning that it will be useful for sequencing from within 7R into the vector sequence of pGPS2.1. We’ll come back to this point in a minute. You can look at Primer S in 7L on your own later. The transposon also includes the CAT gene, which confers resistance to chloramphenicol in bacteria. You’ll select for the chloramphenicol resistance after performing transposition reactions, ensuring that the only plasmids recovered will be those containing the transposon. Notice the duplicate replication origins (R6K). This replication origin is not recognized by standard E. coli strains (like DH5α) and therefore this plasmid cannot replicate in most strains of E. coli. pNIG6, on the other hand, contains a standard colE1 origin, which is recognized by most E. coli strains (including DH5α). After the transposition reaction with pGPS2.1 and pNIG6, the only plasmids that replicate in DH5 and confer resistance to chloramphenicol are ones in which the transposon has hopped into pNIG6. Be sure you understand why unaltered versin of pGPS2.1 or pNIG6 cannot be recovered using this selection scheme.
Performing a transposition reaction with pGPS2.1 in the presence of pNIG6 will generate two types of transposon insertions in pNIG6: either in the vector (pRG3) or in the insert (nimX region). Look at GPS Hop in pNIG6 insert to see an example where the transposition in within the nimX region. Open the component fragments folder and right click on fragment of pGPS2.1 and choose find. Note that the region selected in the graphics pane is in between exon 1 and exon 3. It must have disrupted exon 2 because that feature is no longer displayed. When you recover transposon hops into pNIG6 by selecting for DH5α transformants that are chloramphenicol resistant, you will not know which are hops into the vector and which are into the insert. One way to tell is to digest the clones with PstI and XbaI, which yield a vector band and an insert band. If you isolate a clone where the transposon hopped into the vector, then the insert band size will be unchanged but the vector band will increase in size. If your clone has a hop in the insert, the alternative result will be obtained.
To perform restriction digests and get the predicted results using Vector NTI, pull down the analysis menu, choose restriction analysis, restriction fragments, selecting the enzymes, and click OK. A Restriction fragment folder will appear in the text pane and it will list the sizes of the fragments generated by your digest and will list the sites at the end of each fragment. For example, if you perform this analysis on pNIG6, selecting XbaI and PstI as the enzymes, your restriction fragment folder will list a 4663 bp fragment (from PstI at position 1 to XbaI at position 4664 and a 2496 bp fragment from XbaI at 4663 to PstI at 1). Do this with the GPS Hop into vector and GPS Hop into insert plasmids and compare the results for all three plasmids. This will help prepare you for interpreting your lab results.
If you were to sequence the GPS Hop into insert plasmid using the GPS sequencing primer, Primer N, the resulting sequence would include part of 7R and then the sequence of pNIG6 starting at base 7359 on the bottom strand. Similarly, sequencing with Primer S would yield sequence that started within 7L and then would enter pNIG6 at base 1 on the top strand. Notice that in the GPS Hop into pNIG6 insert example, the sequence data from Primers S and N tell you the exact position the transposon inserted within the nimX gene. You’ll demonstrate your understanding of this point as part of your homework assignment.
HOMEWORK 2: Due by the start of class, Monday November 12th: Assume that you have performed sequencing reactions using Primer N and S on the plasmids GPS Hop into pNIG6 insert and GPS Hop into pNIG6 vector. Also assume that the sequence data you obtain begins with the first nucleotide after each primer and continues for a total of 100 nucleotides. This would yield four DNA sequences, each 100 nucleotides long.
For part (a) of this homework assignment, generate four DNA molecules using Vector NTI, one for each of these four sequences that would be generated by sequencing the “Hop into pNIG6” plasmids with primer S and primer N. Create the sequence files by selecting the 100 bp that correspond to each of these sequences in GPS Hop into pNIG6 insert/vector, copy them using control-C, and use the file menu, create new sequence, using sequence editor function of vector as you did in the first Vector NTI experiment to create the Y306H NsiI DNA molecule. Name the individual sequence files:
“Insert Primer S your name” for the sequence obtained with Primer S using GPS Hop into Insert as template;
“Vector Primer S your name” for the sequence obtained with Primer S using GPS Hop into Vector as template;
“Insert Primer N your name” for the sequence obtained with Primer N using GPS Hop into Insert as template;
“Vector Primer N your name” for the sequence obtained with Primer N using GPS Hop into Vector as template;
Turn these sequence files to me as part of an archive file called GPS (your initials) that contains the four sequence files plus the other molecules you’ll create for part (b).
For part (b) of this homework assignment, use the GPS manual and Vector NTI to determine why the sequences you obtained in part (a) of the homework are not exactly correct. One hint: the sequence files you generated in (a) are incorrect because the sequence of the plasmids, GPS Hop into insert/vector are not exactly correct. A further hint: The GPS Hop into insert/vector plasmid sequences are five base pairs shorter than they should be. For your answer, make new versions of GPS Hop into insert and GPS Hop into vector that are correct. To create the new versions of these plasmids, use the same method you used to create the mutant version of pNIG6 in the Vector NTI experiment and in your first homework assignment. For example, to make the corrected version of the GPS Hop into pNIG6 insert, open it, save it as “GPS Hop into pNIG6 insert your name”, then use the edit function to make whatever changes are necessary. Save these files to disk/flash card and bring them to class. Do both plasmids (hop into vector and hop into insert).
For part (c) of this homework assignment, use the GPS manual and Vector NTI to determine how to use restriction digests to map the approximate location of the transposon hop into the insert of pNIG6. You’ll do this exercise on one of your clones containing a transposon hop into the nimX region of pNIG 6 in the lab. Here, use the plasmid, GPS Hop into pNIG6 insert, and design restriction digests that could be used to locate the position of insertion in pNIG6 and to determine the orientation of the transposon. You’ll need to do two, double digests. Perform the digests using Vector NTI as in the example above. Print the text pane, which will show the folders and the information from the restriction digests, for each of the double digests. Turn a copy of the printout in to me along with parts (a) and (b) and enter this information into your notebook for use when you map the position of your transposon insertion in Experiment 3 in the lab.
Vector NTI Session for Experiment 4: Cre-Lox Recombination and Protein Expression in E. coli:
Each group will use a Cre-Lox-based system to subclone the coding sequence of the nimA gene into a bacterial expression vector derived from pET15b. The vectors we are using were published as part of “The Univector Plasmid Cloning System”, which was designed to facilitate construction of many different plasmid constructs to express the same protein. For example, in experimental molecular and cellular biology, one often wants to express a eukaryotic protein in bacteria to use as an antigen for making antibodies and in eukaryotic cells (insects or yeast) to produce post-translationally modified versions of the proteins. In addition, it is often useful to add multiple different epitopes (epitope-tag) the proteins when expressed in both bacteria and in eukaryotic cells. Because each expression plasmid and each type of tag requires specific cloning strategies, it can take a long time to generate the necessary constructs. The univector system allows one to clone an open reading frame into a single donor plasmid, and then use the Cre-Lox site-specific recombination system to move the open reading frame into each of many Host Expression Plasmids all in an afternoon’s work. In our lab, we’ll just use a single expression plasmid, derived from pET15b, which is used to express proteins that are tagged with the 6-His epitope. We’ll confirm that the correct clone has been made and then will transform the clone a strain of E. coli commonly used for protein expression (BL21 cells). You’ll express the protein in BL21 cells, isolated total protein from those cells, run an SDS-PAGE gel and then detect your recombinant protein by western blot analysis.
Cre is a site-specific recombinase that binds to sites called lox sites. LoxP is the lox site in the plasmids we are using. The loxP sites are 34 bp sequences that comprise 13 bp inverted repeats around a central GCATACAT core. Thus, loxP sites are directional, and Cre-mediated recombination always occurs with the loxP sites oriented in the same direction. The loxP sequence also has an open reading frame that is in frame with the ATG of all Host Expression Plasmids. Thus, with the Univector system, you clone your ORF downstream of and in frame with the donor plasmid (pUNI10) loxP sequence, and use Cre to catalyze recombination with a Host Expression plasmid, in which loxP is downstream of a promoter and translation initiation signals. Recombination fuses the two plasmids, placing your ORF downstream of the promoter and translation initiation signals of the Host Expression vector and in frame with the ATG. Let’s use Vector to go over these details, using the plasmids you’ll use in the lab. Open the molecules, pAS4-16, pHB3-HIS6 new, and pAS4-16 plus pHB3-HIS6 new (three plasmids).
pAS4-16 is the pUNI10 donor vector with the the gene nimA’s ORF cloned as a NcoI to SalI fragment that is in frame with the loxP coding sequence. Confirm this by opening the Component Fragements folder, right clicking each component and using the find component fragment function to highlight pUNI10 and then the nimA fragment. Selecting the nimA coding sequence in the graphics pane, which is the arrow on the plasmid in the graphics pane, will highlight the coding sequence in the sequence pane, which you will see starts with an ATG at the NcoI site. This is in frame with the loxP reading frame, which you can illustrate by selecting from the start of loxP (2121) through the NcoI site (2189) and using the analysis menu to translate this sequence in the sequence pane on the direct strand. The translation shows that the loxP ORF, which is ITSYSIHYTKL, is in the same, uninterrupted reading frame as the ATG in the NcoI restriction site.
Now look at pHB3-HIS6 new. Note that the direction of transcription and translation in this expression vector is right to left on the map and sequence pane (the promoter is labeled T7 Promoter). Click on the NcoI site in the graphics pane, which contains the ATG start codon on the bottom strand. To see that this ATG is also in frame with the loxP site, select the sequence from the loxP sequence (position 5709) through the ATG (position 63) and pull down Analysis, Translation, In Sequence Pane, Complementary Strand to translate the sequence and show the translation in the sequence pane. Note that the loxP ORF reads ITSYSIHYTKL (same as in pAS4-16) and that this is in the same, uninterrupted reading frame as the ATG at the NcoI restriction site. So, nimA is in frame with loxP in pAS4-16 and loxP is in frame with the ATG in the host expression vector, pHB3-HIS6.
pAS4-16 and pHB3-HIS6 new were combined by site-specific recombination involving the loxP sequences on both plasmids using Cre Recombinase in vitro. Look at the resulting plasmid, called pAS4-16 plus pHB3-HIS6 new. This map is drawn to orient transcription and translation of the NIMA::HIS6 fusion from left to right as is the normal convention. The component fragments are both complete plasmids so the combined plasmid has two loxP sequences, one in between the promoter and the nimA ORF (which straddles sequence position 1), and the other at the end of the pAS4-16 component (at 4429). Transcription by T7 polymerase produces an mRNA that will be translated using the the ATG at 10092 through the stop codon at 2150. Remember, the goal of all this is to produce a fusion protein containing the nimA protein fused to a six histidine (HIS6) epitope. To check this, translate the sequence you’ve selected. The fifth amino acid in the translation is the first of the 6 histidines of this epitope tag (so far, so good). The translated frame continues uninterrupted through the nimA ATG at position 51 and, of course, all the way through the nimA cDNA sequence (correct!).
Let’s make a protein molecule that will provide information on the predicted fusion protein, including its size and charge. With 10092 to 2150 still selected, pull down Analysis, choose Translation, choose Into New Protein, and Direct Strand. A box will open suggesting a name for the protein molecule. Add your group number or initials to the name and click OK. This sequence will now be present in a protein molecule file in the Protein Database. In the text pane, open the Analysis folder, and you will find information such as the predicted molecular weight and the proteins isoelectric point (the greater than pH9 isoelectric point of nimA is unusual and could be used to rapidly purify this protein by ion exchange chromatography). The MW is a little over 83 kD, so you’ll look in this region of your gel and western blot for evidence of expression.
HOMEWORK 3:
Due by the start of class Monday November 26th:
Part (a) of your homework will help you prepare for analysis of clones you create using Cre-mediated recombination on pAS4-16 plus pHB3-HIS6. Use Vector NTI to predict the results of a PstI digest of the correct fusion plasmid. Record this information in your notebook for use in the lab and hand it in as part of your homework assignment.
For part (b) of your homework, create a Univector host expression plasmid using Vector NTI that could be used to express a HIS6-tagged protein in Aspergillus under control of the regulatable alcA promoter. alcA encodes the enzyme alcohol dehydrogenase and its transcription is tightly regulated in Aspergillus. Transcription of alcA if very low in cells cultured in medium containing glucose and is induced over 100 fold when glucose is removed and replaced by ethanol. Use the plasmids, pAL4 plasmid and pHB3-HIS6 mod to create such a plasmid. pAL4 has almost everything needed (the alcA promoter, the alcA ATG, and selectable marker for transformation of Aspergillus to uracil prototrophy (Nc pyr4). It does not have a loxP sequence and the six histidine codons, both of which can be obtained from pHB3-HIS6 mod. Select the sequences you need from each sequence and put them in the fragment goal list in the correct orientation and then open the fragment goal list, choose run, and give the new plasmid the name pUNI alcA plus your last name.
Vector NTI Session
for Experiment 5: Gene Knockouts
You will be performing a Gene Knock-out experiment in Aspergillus nidulans. You will be deleting the UBC11 gene and replacing it with a selectable marker (the Af pyrG gene). You will accomplish this by first, constructing a linear DNA fragment that is homologous to the DNA regions that flank the UBC11 gene, which contains the Aspergillus fumigatus pyrG gene in place of UBC11.
You’ll construct this DNA molecule by “fusion PCR” using the strategy diagramed in the top, left figure on the last page of this handout. A related strategy can be used to “epitope-tag” the gene (shown in the top right figure, same page). The rationale for using this strategy is to generate constructs suitable for gene deletions (aka knockouts) or tagging (knock-ins) rapidly without having to clone them. 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 animAl genome manipulations. The strategy for deletions involves generating ~1 kb PCR fragments corresponding to regions that flank the gene of interest. Each flanking fragment has 21 bp sequence at one end that is identical to the sequence at one end of a PCR product that contains a selectable marker (the Af pyrG gene, referred to as the Af pyrG Cassette). PCR amplification of all three fragments as templeate (5’ flanking region, Af pyrG Cassette, 3’ flanking region) using primers that hybridize near the ends of the flanking fragments results in formation of a ~ 4 kb amplicon corresponding to the fusion of all three template fragments.
The deletion construct will be transformed into A. nidulans, where it will replace the endogenous UBC11 locus by homologous recombination (bottom left figure, last page), resulting in a deletion of UBC11 and an insertion of the Af pyrG gene in it’s place. Selection for Af pyrG (growth in the absence of uracil) will identify transformants that contain the PCR fusion product integrated somewhere in the genome. PCR will be used to screen for those transformants in which homologous recombination occurred (bottom right figure, last page). Note that all transformants will yield a PCR product using the 5’ For and 3’ Rev primers that amplify the Af pyrG gene, whereas only transformants generated by homologous recombination at the UBC11 locus will yield products with the primer combinations, P1 + 3’ Rev or 5’ For + P6.
A key reagent in this procedure is the A. nidulans strain called TNO2A7, which has a mutation in a gene (nkuA) required for Non-Homologous End-Joining (NHEJ). NHEJ is the major pathway used by most eukaryotic cells to repair dsDNA breaks and appears to be the major pathway by which transforming DNA gets inserted into a chromosome. The NHEJ pathway also acts on exogenous DNA added to cells during transformation, and inserts the DNA fragments randomly into the genome, making the frequency of insertion by homologous recombination low. In the absence of NHEJ, A. nidulans now acts like yeast and uses primarily homologous recombination. Thus, one can screen a few transformants by PCR with confidence that most/all of them will be gene knockouts/knockins (Nayak et al., 2006). Mutation of NHEJ genes has been shown to be a generally applicable in filamentous fungi to increase the frequency of homologous recombination and might be applicable to higher organisms as well.
We will use Vector NTI to design PCR primers that could be used to generate a fusion PCR product for deleting UBC11. The primer terminology and strategy is as indicated in the figures on page the last page of the Experiment 5 handout. To use Vector NTI to design PCR primers, you must first choose a region of a DNA molecule to amplify. Open AN5495 UBC11, which is a Vector NTI molecule representing 12 kb of the A. nidulans genome, including the annotated gene named AN5495.3, which is the putative Aspergillus ortholog of UBC11. First, we’ll design primers to amplify the 5’ flanking region. Select the UBC11 CDS using the graphics pane, 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 and the box to the right indicates the same for searching to the right of the selection. Type 100 in each of these boxes (it can pick primers anywhere in these 100 bp regions). 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, the Probe Conc should be 250 pMol, and the dG temperature should be 25 degrees. The TM should be between 55 and 65 degrees. The %GC should be between 40 and 60. The length should be between 18 and 23. The pimers 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. This amplicon is now highlighted in the
graphics and sequence panes. It should
start at 5358 and end at 6404. The
primers are each 21 nucleotides long with Tm close to 55 degrees. Save this PCR product as UBC11 5’ flank (your
group number) 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, do so and click OK. The new molecule file will open. The component fragment folder indicates where
this came from. The primers are features
that we will save as oligonucleotides in our database. Do this by selecting the sense primer using
the text pane, make sure the sequence pane is active, click the Add to Oligo
List icon, name the primer UBC11 P1 (your group number), 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. To save these oligos to the
database, open the oligo list by clicking on the Open Oligo List icon, choose
the P1 oligo, click save, choose the BIO 510 2007 subset, and click OK. Do the same for P3.
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), pull down the analysis mneu…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 degress, so this is fine. Right click the product, save to database and open window, name it UBC11 3’ flank (your group number), click OK, BIO 510 2007, 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 P4 (your group number), and the antisense primer should be UBC11 P6 (your group number). 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. Open the oligo list, select UCB11 P4, click save, BIO 510 2007, OK. Same for UBC11 P6.
Now you have primers that are useful for amplifying flanking regions, but the flanks will not have homology to the Af pyrG Cassette. To amplify 5’ and 3’ flanks with the appropriate homology to the Cassette, specific 21 nucleotide sequences need to be added to the 5’ end of P3 and P4. The 21 nucleotides to be added to the 5’ end of P3 correspond to the 21 nucleotide sequence of the bottom strand at the left end of the Af pyrG cassette. This sequence is in the oligo file, “5’ end of P3’s for Af pyrG cassette”. Open that oligo file by doubling clicking, copy the sequence cntrl-C, click cancel to close the box, open your UBC11 P3 oligo, click to the left of the sequence in the sequence box to put the cursor at the 5’ end of this sequence, paste cntrl-V, click OK to save the sequence. Now add the “5’ end of P4’s for Af pyrG Cassette” to the 5’ end of your UBC11 P4 seqeunce in a similar manner (open the 5’ end of.. oligo, copy, cancel, open your P4, click to the left to add to the 5’ end, paste, OK to save).
There are two primers remaining to be designed; P2 and P4. These are “nested” primers that will be used in the fusion PCR reaction, where you use the 5’ flank, the Cassette, and the 3’ flank as templates. They should prime synthesis just inside the P1 and P6 primer binding sites. To generate these primers, we first need to make a new molecule that is the combination of the UBC11 5’ flank, the Af pyrG Cassette, and the UBC11 3’ flank. Open all three files, put the entire 5’ flank sequence into the fragment goal list, put the Af pyrG Cassette in the list, and then put the 3’ flank in there. Open the fragment goal list, click run, name the file UBC11 P1 to P6 (group number), CLICK LINEAR, then construct, into BIO 510 2007, OK. This will save the file in the database and open a window showing the file. Close the fragment goal list to save space. Before using this file, make sure it’s correct. First, look at the components in the text pane. Should be 5’ flank, then cassette, then 3’ flank. Second, check by adding the PCR primers P1, P3, P4 and P6 to the motifs in the analysis menu and make sure they hybridize where they are supposed to hybridize (the correct location and the correct strand), and make sure they match 100%. 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 end of the 5’ flank and the beginning of the Af pyrG Cassette, whereas the P4 primer should match the end of the Af pyrG Cassette and the beginning of the 3’ flank.
Now we can design primers to amplify this molecule. Select from 101 to to 4004, anslysis…primer design…amplify selected…OK. The first product 3949 bp from 100 to 4048, will work just fine. Save it into the database and name it UBC11 P2 to P5 (group number). 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.
Note: the primers you will use in this lab exercise were designed previously and correspond to the UBC11 oligos and DNA molecules in your database, and not necessarily to the oligos you just designed. Look at the files of 5’ flank, 3’ flank, P1 to P6, and P2 to P5 to determine what you should expect to see in gels of your PCR reactions.
HOMEWORK 4:
Due by the start of class Friday December 7th:
Part (a) Design PCR primers to create a deletion construct of the A. nidulans gene, AN2761 UBC4. You have a Vector NTI file of the Aspergillus genomic region containing this gene and the Af pyrG PCR Cassette in your database. Save the 5’ flank, 3’ flank, P1 to P6, and P2 to P5 DNA molecules (name them UBC4…(your initials). Save the primers as UBC4 P1 (your initials), P2 (your initials),…P6 (your initias). Save a copy of these files as archive files and turn them in by Friday, December 7, start of class..
Part (b) Determine the size of the PCR products that will be detected during analysis of an Aspergillus transformant that was generated by homologous recombination of the UBC11 del P2 to P5 fusion product we generated in lab. To do this, select the entire UBC11 del P1 to P6 fragment and use the Anaysis, Primer Design, PCR Using Existing Oligos to generate three PCR poducts: 1) using primer pairs UBC11 del P1 + 3’ Reverse Af pyrG Cassette; 2) using primer pairs 5’ Forward Af pyrG Cassette + 3’ Reverse Af pyrG Cassette; 3) using primer pairs 5’ Forward Af pyrG Cassette and UBC11 del P6 (that’s three different PCR reactions). Save each PCR product as a file (1 = UBC11 del P1 to 3’ your initials Rev; 2 = UBC11 5’ For to 3’ Rev your initials; 3 = UBC11 5’ For to P6 your initials). Include these files in the archive you created for (a) and turn them in by the start of class of Friday.