Bacteriophage Evolution Lab Week

Transcription

1 Bacteriophage Evolution Lab Week Introduction Items you must bring to this lab: 1. Write-up from Phage Lab 5 2. The number and letter of the original phage suspension (1A-16A, 1B-16B) that your sequences came from. Note: If you switched over to using another group s sample at any point, you need to have the number of that group s phage suspension. Some groups switched because of not getting phage at 20 degrees, or not having PCR reactions work. In this lab you will do what might be a first step in data analysis for someone doing this type of research. You will be determining if you have a mutation(s) in your phage (in the regions sequenced) and looking for where those mutations are located as a first step toward searching for clues as to why any mutations might have given rise to the ability to grow at reduced temperature. If your mutation has resulted in an amino acid change in protein F or protein D, you will be using a three-dimensional protein visualization program to see where that altered amino acid is likely to lie within the phage procapsid (immature phage particle) or capsid (mature phage particle). The locations (in different mutants) of protein changes within the 3-D structure of the viral particles may help the entire class formulate hypotheses about the nature of beneficial mutations that allow these types of phage to evolve to growth under new conditions. Protein F is the main phage capsid protein (the capsid is the mature phage particle). Protein D is the outer scaffold protein, which helps assemble the phage procapsid, but which falls off the procapsid as the last step in formation of the capsid. It will help you during this lab to have the Phage Lab 5 write-up so that you can refer to phage proteins, phage assembly, and structural figures there. You will use two extremely powerful state-of-the-art DNA/protein software packages in this lab, VectorNTI and Chimera. Some of the steps you will be performing require surprising amounts of computer time, so there may be steps in which you need to be a bit patient and let the computers crunch. You will use VectorNTI to look at the chromatograms from your sequencing reactions, proofread the sequences, and save the adjusted, good regions of sequence. You will also use this package in a BLAST search to determine where any mutations are located, and if you find any, which amino acid(s) in which proteins have been changed. The BLAST search scans the world s largest DNA sequence database to determine the similarity of your sequence to any sequences within that database. You will use Chimera to visualize the phage procapsid in three-dimensions and map your mutation into the 3-D structure. If you do not find any mutations in your phage, we will assign you another mutation to analyze. Note that the absence of any gene F or D mutation in a phage that can now grow at reduced temperature, is still valuable information. Before this class worked on this project, about 10 mutants of this type had been examined in detail and all had mutations in genes F or D, but there may be other, still unknown routes to the ability to grow at 20 degrees. 1

2 To hand in at the end of this lab: You will print two copies of each of the following, one copy to keep and one to hand in to your TA at the end of the lab. The copies you keep will be essential for writing up your research paper. 1. A print-out of your sequence aligned with ID8 identifying any base change in your sequence. Write down the change on this sheet. For example, if ID8 has a T at base 3507 and your mutant has a C there: 3507, T C. Also, write down the amino acid and codon substitutions: 52, S T, etc. On this print-out write the original phage suspension that your sequence came from, your name, and your group s initials. Do this even if you found no mutation in your sequences. 2. A print-out of your phage structure, showing the location within that structure of your amino acid change. If you found no mutation, then print out the structure with the mutation you were assigned for today s lab and indicate this on the figure. 2

3 DNA Sequence Analysis Tutorial Genetics Laboratory By Jason Evans, Luke Sheneman and Celeste Brown 1. What is VectorNTI? From the Informax website: VectorNTI equips laboratories with an extensive range of software tools for sequence analysis and molecule manipulation. Integration of data and analysis tools is achieved using an intuitive, object-oriented database for the storage and organization of DNA, protein and oligonucleotide sequences, and other molecular biology data. From the Database Explorer, users can launch comprehensive, publication-quality views of any sequence in an integrated, multi-pane Molecule Display window. Within this window, make a selection of some sequence graphically--an Open Reading Frame (ORF), for example--and apply all the tools necessary to design multiple sets of PCR primers, initiate a BLAST search against any division of GenBank, translate using any one of numerous genetic codes, or automatically design a cloning strategy to create a new recombinant molecule. The VectorNTI Local Database stores any new protein sequences generated, or PCR primers designed, and keeps a record of the parentdescendant relationships that might exist between molecules. These workflows can be performed without any reformatting of data, allowing the researcher to concentrate on the science, rather than the technology, underlying their bioinformatics analyses. Many additional bioinformatics workflows are possible within Vector NTI. Molecular biology data management: storage of DNA/RNA Molecules, Protein Molecules, Enzymes, Oligos, Gel Markers, Citations, BLAST Results and Analysis Results Creating, mapping, analyzing, annotating and illustrating DNA and protein sequences PCR, sequencing and hybridization primer/probe design BLAST searching, viewing of results, and recovery of hits Recombinant cloning strategy design PubMed/Entrez searching, viewing and recovery of results In silico gel electrophoresis Connectivity to numerous Internet analysis tools Primer Design DNA Sequence Analysis Protein Sequence Analysis BLAST Searching Database Explorer 3

4 2. Goal The purpose of this exercise is to locate the mutations derived from your evolution experiments, visualize them in the structure of the procapsid and consider how these mutations may help the phage grow at 20 C. 3. Getting Started Start Windows XP by double clicking on the icon on the desktop. Double click on the Vector NTI Explorer icon that is on the left hand side of the Windows XP screen. 4. Using ContigExpress We will use ContigExpress to create a consensus DNA sequence from the raw chromatograph data produced by the automated DNA sequencer. ContigExpress is a tool that allows you to assemble DNA fragments, both text sequences and raw chromatograms directly from an automated DNA sequencer, into overlapping sequences known as contigs. Users can create ContigExpress projects and store sequences, contigs, and the options used when assembling the contigs. In order to create a contig from distinct DNA fragments, you will learn how to Create and manipulate a ContigExpress project Assemble contigs Edit a contig to remove ambiguities Export a contig as a single DNA sequence for further analysis 4.1. Click on Assemble in the top menu bar of Vector NTI window, then select ContigExpress. The ContigExpress Project window will open At this point, you need to get your sequencing results In the top left hand corner of the XP window, there is a folder marked MacOS desktop. Double click on this folder, then on the folder named DATA. Open the Bio210 folder, and then the folder for your lab section Select your group s ab1 files and drag them to the Fragments(MAIN) folder in the ContigExpress Project window. You may get a message asking if you want to take the labels from the file names or from the internal definitions: do NOT use internal names Once you drop the fragments into ContigExpress, you should get a message indicating that the fragment importing was successful. Click on OK. Close the folder from which you retrieved your abi files. 4

5 4.3. Assembling DNA fragments essentially performs an alignment of the overlapping regions of the fragments. Select all of your fragments inside the ContigExpress project window. Select Assemble Assemble Selected Fragments from the ContigExpress pull-down menu. The program tells you how many contigs were assembled and how long it took. Click on OK Your contigs are in Assembly 1. Rename the Assembly by clicking on Assembly 1 and then waiting a second and clicking again. Change the name to reflect your Class Period and Name (eg. TuAM_CBrown). You can rename your Contigs and your individual fragments if you like using the same procedure. 5

6 4.5. Now double-click on your contig (i.e. Contig 1 ) in order to edit it. A contig editor window will open. This window is broken up into three main panes. The upper left pane contains simple information about the contig, a description of the contig, and a description of the fragments within the contig. The upper right pane presents a graphical representation of the contig and the way in which the fragments overlap in the contig. Also shown in the lower portion of this pane is the location of known discrepancies in the contig. These discrepancies represent nucleotides that differ between the two sequences, and will need to be resolved. The lower pane depicts the actual sequences themselves and depicts the fundamental details of the contig, showing the exact way in which the contig was constructed from the sequence fragments. Ambiguities and gaps are presented here in detail, and can be resolved manually in this lower pane You can visualize each chromatogram from which the sequences were derived by rightclicking anywhere in the lower pane and selecting Show All Chromatograms from the pop-up window. Viewing the chromatograms will help you resolve discrepancies and determine where your sequence is good enough to use in future analyses. Remove the ends of the sequences where the chromatograms are poor (overlapping, low or broad peaks) Correct discrepancies using the following procedures. Locate all ambiguity codes (possibly highlighted in yellow in the consensus sequence (Contig 1) at the bottom of the lower pane, as a symbol underneath the consensus sequence and as green vertical lines in the graphics pane) and resolve the ambiguity character with the most likely alternative. In most cases, the most appropriate solution is simply to choose the unambiguous code from the other sequenced fragment in the same aligned column (where it overlaps). 6

7 Locate and remove all gaps. In some cases, the solution here is to remove the entire column, while sometimes the solution is to change the gap code in one of the fragments to the most obvious alternative character. Use the chromatogram as your guide. Also, choose the better chromatogram. In the picture above, the top chromatogram is good and the lower chromatogram is very poor. Action How to Perform Sequence Pane Result Chromatogram Pane Result Delete Select residues; ( ) replaces NTs; Nts ( ) appear in upper sequence press Delete moved below strand Insert Place caret; type new Nts ( ) appears below new NTs; new NTs are colored A break appears in the chromatogram Replace Select NTs; type new Nts New NTs appear in strand; replaced NTs moved below strand New NTs appear in upper sequence; no break in chromatogram 5. Save Contigs as standalone DNA sequence 5.1. Once all ambiguities are resolved, select (highlight) the entire consensus sequence. The easiest way to do this is to right click on Contig1 in the bottom pane and select Select All. Right click on the consensus sequence and select Copy sequence from 1 bp to n bp. 7

8 5.2. At this point, the consensus sequence representing the assembled contig of the DNA fragments is in your cut/paste buffer. Go back to the Exploring-Local VectorNTI Database. Select Table New Molecule (Using Sequence Editor) A dialog pops up which allows you to annotate, name, and edit the characteristics of the new molecule In the General tab, give the new molecule a name that reflects your lab Day and Time, your Name and the sequence number (Tu_AM_CBrown_1). Next, click on the DNA/RNA Molecule tab and make sure that the DNA is marked as linear and that the molecule is treated as DNA rather than RNA Next, click on the Sequence and Maps tab in this dialog, and then click on the Edit Sequence button in this dialog A dialog box will open allowing you to enter the complete DNA sequence of the consensus contig. Paste the consensus contig sequence into this window by clicking on the Paste button, and then pressing OK. Then click on the Comments tab and add any relevant information for your sequence. Press OK You should now have a linear DNA sequence that represents the fully assembled DNA sequence open in a window. Repeat this process with each contig or singleton sequence so that you get a file for those sequences as well. 8

9 6. Compare your sequences to a nucleotide sequence database You will use the BLAST (Basic Local Alignment Search Tool) program to identify the sequence in a database that is most closely related to your sequence. The database you will search is Genbank, the nucleotide sequence database (nr) at the National Center for Biotechnology Information at the National Library of Medicine in Bethesda, Md. The results will show you up to 500 sequences that are similar to your sequence. The first sequence in the list is the sequence in which you are interested Open one of your sequence files. From this window, perform the BLAST search by selecting Tools BLAST Search. You will see two screens, just click OK for each The default settings for the BLAST program are exactly what you would like to use, so press the Submit button The status column tells you what the program is doing. After awhile, the BLAST search will finish. Double-click on the name of YOUR sequence to review the results of the BLAST search To see the hits from the BLAST search, expand the hits folder in the leftmost pane in the BLAST Results Viewer window by clicking on the + : Select the name of the first molecule in the Hits list. NOTE whether your sequence is in the same direction (direct) as the BLAST result, or on the complementary strand by looking at the Graphics Pane. You will see an alignment of your sequence and the BLAST hit in the bottom alignment pane. Select the Alignment Pane by clicking on the second to the last icon on the Active Pane: line Print 2 copies of the alignment by selecting BLAST Results -> Print. You will use this print out to identify the location of mutations in the ID8 genomic sequence, which you will download next. Be sure to pick up your alignment and not someone else s; there is NO unique identifier on each page Click on the blue underlined link for the first hit. This action will download the genome sequence for ID8 to your computer. Select FILE and Save As and save the file to the DNA/RNA database. 9

10 Repeat the BLAST search with each of your sequences. Write down the number indicating where your sequence differs from the ID8 sequence. DO NOT repeat step 6.2.3, you only need to download the ID8 sequence once. 7. Identify mutations in your sequence 7.1. Now, find the region that matched your sequence in the ID8 genome sequence Using your alignment printout, identify the ID8 position number of the nucleotide that differs between your sequence and the ID8 genomic sequence Click on the long box on the bottom edge of the Vector NTI window of the ID8 genome sequence. A dialogue box will open into which you should type the nucleotide position determined in step When you click OK, the selected nucleotide will be highlighted in the sequence pane and outlined in the graphics pane. Notice which gene has the mutation. (If you have more than one mutation, you will need to do the following procedure for each gene.) 10

11 7.2. Translate the sequence of the ID8 gene so you can see what amino acid was affected. Remember you are interested in D and F, go back and do mutations in C, A and B if you have time Your sequence should be highlighted in the sequence pane. Find the icon at the top of the screen that has an M over an arrow over ATG. Click on this icon Use the Edit -> Find Sequence function to locate the nucleotide position of the mutation If your sequence is the complement of the ID8 sequence, be sure to check that box option in the dialogue. Type in ten bases of ID8 sequence preceding your mutation from the alignment, and click on Find Next Write down the amino acid that is affected by the mutation and the codon that encodes it Go to Analysis->Translation->Set Genetic Code, and determine what altered amino acid your mutant has at this site. Don t close this window, you will need it again Before you go on to the final step, you must find the position of the amino acid that has changed in your protein sequence Be sure that the gene you wish to translate is highlighted in the graphics pane Right click on the highlighted gene, then select Translate -> Feature into New Protein. This will produce a protein that you will save in the Protein Database with an appropriate name Again, find the amino acid position of your mutant by using Edit -> Find Sequence. You will need to translate the 3-letter amino acid names with the single letter names using the Genetic Code window from Close all of the Vector NTI windows. Then go to Start -> LOGOFF. When the login screen comes up, click on Virtual PC and the Quit. 8. Identify mutations in your structure Now you will find the mutations in the structure of the phix procapsid. Identifying the locations of the structures may help determine how these mutations help the phage to adapt to the conditions of the selection experiment Open the Biology Applications folder that is on the Desktop, and double click on Chimera to start it Go to File -> Fetch by ID and type 1CD3in the box next to PDB ID. Click on Fetch When the structure opens, drag the bottom right hand corner of the frame to enlarge the picture to about ¾ of the screen. This structure is one fifth of a pentamer, and 12 pentamers make a complete phage procapsid like ID8. Click and drag to rotate the structure Use Select -> Chain -> B to select the B protein in the structure Use Actions -> Color, to color the B protein red Repeat 8.4 and 8.5 to color proteins F blue and G yellow. When you are finished, select Select -> Clear Selection. The structures that are still white are the 4 D proteins. 11

12 8.7. To remove the side chains and view just the backbone structure, Select Actions -> Atoms/Bonds -> backbone only. Rotate this structure by holding down and moving the mouse, you should be able to clearly see the alpha helices in F and the beta sheets in G. The straight line in B is an incorrect representation of sequence for which there is no structure Now you will view the procapsid. Select Tools -> Higher Order Structure -> Multiscale Models. Drag this window onto the desktop so you can see the whole structure window. Click on the button Make models, which is at the bottom of this menu In a short time, the procapsid structure will form. Right click on the structure and move the mouse away from you to make the figure smaller Choose Tools -> Viewing Controls -> Sideview. Move the camera (little yellow box) and clipping (yellow lines) to see how they affect your structure. You should be able to reproduce the structure shown here. Move the left clipping lines to the left of the structure so that you can see the surface of the subunits. Turn the molecule so that your figure matches the orientation of the downloaded structures. The pale blue star in the center is the G protein pentamer. The purple structures underneath are the F proteins. All of the other subunits that you see are the D proteins that form the outer scaffold. Your colors may vary Now move the clipping lines to replicate this structure that shows the inside of the capsid. The green structures surrounded by purple are the B protein End your explorations by putting the left clipping line to the left of the structure and the right clipping line to the middle of the structure. This will help when viewing the wire structures in the following steps. Also, move the little yellow box so your procapsid fills the whole screen Select the entire procapsid by holding down the control key, left clicking in the top left corner of the window and 12

13 moving to the bottom right corner. This will outline the entire procapsid in bright green In the Multiscale Model window, click on Show which is next to Style. In the new menu, select Wire. Then WAIT until all subunits have changed, this will take several minutes. You should see each subunit change one by one Lets get rid of the D proteins. Click on Select -> Chain -> 1. It will take about a minute for all of the chains to be selected, be patient. The Select button will be boxed while the program is doing this task. Wait until the box goes away before proceeding Click on Actions -> Atoms/Bonds -> Hide. Again, be patient, it will take about a minute for the chains to disappear Repeat 8.15 and 8.16 for Chains 2, 3 and 4. When you are done, you should be able to clearly detect the capsid structure Let s color the subunits so that all the G s are blue, all the F s are white and all the B s are cyan by repeating what you did in steps Remember that B is on the inside of the structure, and you may not see anything happen during the Select or Action phases. Your structure should look like the picture to the right Now you need to find the location of the amino acids in the F protein that changed in your experiment. Choose Tools -> General Controls -> Command Line In the window at the bottom of the screen, you will select the amino acid that you wish to locate in the structure. For protein F only, the actual amino acid number is one less than what you see in the alignment (the starting Methionine is NOT present in the structure). If your mutant was at amino acid 419 of structure protein F, you will type in sel #*:419.F. Then hit the return (enter) key. This will not take as long as selecting whole proteins. Notice that the number of atoms that are selected is listed at the bottom of the screen Change the color of these selected atoms to red Choose Actions -> Atoms/Bonds -> Sphere. When the Action button is no longer boxed, make the structure bigger until you can see what these atoms look like. It is hard to see what is going on in the spaghetti, but manipulate the structure until you have a pretty good feel for where the molecule is. Go to 8.25 to learn how to print a picture of the structure. 13

14 8.23. It may help to look only at the backbone. Use Select -> Select All. When the whole structure is outlined in green, go to Action -> Atoms/Bonds -> backbone only. This will bring back the D proteins, but you will need them next anyway. Is your F mutant interacting with the D protein? With G or B? Now visualize your D mutation using the same method outlined in 20. Remember that the D proteins are listed as 1, 2, 3, and 4, so your mutation would be typed in sel #*:45.1. Color these molecules, then make them spheres. The process must be repeated for subunits 2, 3 and 4. Once you have these mutations visualized, turn the structure until you can detect the mutant s interactions with other subunits In order to print the structure with your mutants highlighted, pick a view that seems informative. Then hold down the apple key, the shift key and then type 4. Your cursor will turn into a crosshair. Move the crosshair to any part of the window with your structure, and hit the space bar. The cursor will turn into a little camera, and the Chimera window will be highlighted in blue. Click on the structure window, and a copy of your structure will appear on the desktop. Double click on this picture, and it will open in the program Preview. Print two copies of this picture. Be sure to pick up YOUR pictures. Be sure to LABEL your printouts with your name and other information described in the intro to this lab before handing them in. 14