The Aptamer Database

As a potential off bench project, I mentioned updating the aptamer database. The original publication should be read first to figure out what the database is all about. It hasn't been updated in nearly 4 years and is missing a wealth of current journals. A former member of the Aptamer Stream began updating it, but did not send me her final product and I've lost contact with her, so I guess we should start over again. Aptamer literature is fairly easy to search for on PubMed, but much more difficult to find exactly what you are looking for so having a database of all things aptamer is useful.

For an idea of what we can do with an up-to-date database, check out this article. Basically, we can mine the database for all sorts of information that isn't readily apparent by cursory glance. For instance, we could tell what the most common selection buffer is, or we could look for the most common 4 nucleotide sequence or the most common random region that produces aptamers. We could also find out if a skewed pool or random pool is more beneficial for screening aptamers.

Any of you can begin this project at any time. The first step is to get articles that aren't in the database. To begin finding primary articles, I would first suggest looking at a few of the reviews I've given you. In them are tables of selected aptamers with references. Look up those selections on pubmed and download the articles into Mendeley. Then you should find articles where people have used aptamers and find out the original aptamer publication for the particular aptamer. We are interested in the original selections of aptamers. The database does not include reviews, applications, patents etc. We may look to add these in the future. If you are off campus, you must append the following proxy address before the article: http://ezproxy.lib.utexas.edu/login?url= thus an article from NAR for instance would be: http://ezproxy.lib.utexas.edu/login?url=http://pubs.acs.org/doi/full/10.1021/bi1016233

I have created a Mendeley account for this purpose. Download Mendeley Desktop and use the account aptamer.streamut@gmail.com with the room number as the password. This way, we can keep the collection of pdf files synced up. It is easy to incorporate articles you find in the web using the Mendeley Bookmarklet. Just remember you have to be signed in and turn off your popup blocker for these sites. Then within Mendeley desktop if you double click the article it will download the PDF. I'm not a fan of Mendeley for PDF organization though after using Papers.app for Mac. In searching, a similar option is available for Windows called "I, Librarian". Haven't used or installed, but it seems cross platform and merges the best of Papers with the online collaboration of Mendeley. Sound off in the comments if you like I, Librarian.

Every time you go into Mendeley, sync with the server and then start searching for new stuffs. The sycn makes sure that anyone working on this project will have the most up to date links possible.

As for the database itself, you can and and modify citations from the website directly with the proper credentials. The database is set up like figure 1. Alternatively, we can grant you special access to the sandbox database through MySQL Admin, a graphical front end will be installed through phpMyAdmin, a graphical front end like MS Access that interfaces with the actual database and runs on your computer.

I have also created a webform in Google that allows you to add effectively add information for curation later.

Figure 1. Database table layout

If you find that the information should be displayed differently or that more information should be included, then feel free to tell me. For instance, there is another aptamer database called RiboaptDB and discussed in in this PDF. You may find they do things better or different than we do. Whereas they copied a lot from us, we may find they have implemented information searching or viewing slightly different that makes it easier to use. We can certainly re implement our site better or completely differently so please be aware.

You should also learn a bit about database setup. The aptamer database is hosted from MySQL on a linux virtual machine in the Ellington Lab. You should probably understand a bit about relational databased such as MySQL, so here is a nice tutorial from About.com. I have a complete copy of the database that can be imported into Microsoft Access if you want to play around with your own ways of adding information (i.e. making a new form or something).

Lastly, the project should include some type of website redesign and database input redesign. Using mendeley you should realize that Mendeley uses a database in the back end. If we can create some type of "referenced input" such as BibTeX or RIS format, we wouldn't have to input all the citation information manually. Additionally, if you know web programming (or know of someone who does) and want to try your hand at redesigning the site, please let me know. You could even interface it with some of the blogs or content management systems that are available if you know how to program PHP code. I can get you started on a scratch site to play around with before changes are moved over to the real site.

Tasks

1. Find new literature - Articles detailing the original selection of aptamer binding species. You can also include different techniques, reviews or applications/uses of that aptamer in therapeutic/diagnostic capacity, but separate those into different folders in Medeley. We just want the aptamer sequence for now and the conditions it was selected under.
2. Input all literature into Mendeley - You should label/organize into original selections, other catagories. We don't care much about the potential application for now. We also don't care much about
3. Transfer literature from Mendeley to Database - probably an XML file of some sort.
4. Add sequence information into database
5. Redesign input method
6. Redesign website
7. Do cool things with the data

APP is in

APP came in today and is located in the FedEx box in my drawer. It needs to be resuspended, aliquoted and stored.

Proposal Information

Your target proposals are due today. Originally I had the date set as Tuesday, but I extended the due date.

You should have all the information you need to submit a winning proposal now. The original instructions for the proposal have been posted on Blackboard under Course Documents. When working on it, please take into account the comments and thoughts provided from members of the lab associated with your APS abstract.

For submission, I would like you to learn and use DropBox or WebSpace and provide a link from your APS abstract to the digital file of the proposal (in PDF or Word format). This will keep everything neat and prevent you from having to write something that long into the blog proper. DO NOT MAKE A NEW POST. Find your abstract, edit it with a line near the top that says something like, "The full proposal has been posted on DropBox" and link "posted on DropBox" with the link to your actual file. With the file in the cloud, any changes you make to it I will be able to see in real time. This also allows you to modify and edit the file without changing the link and I will always see the most up to date file. The same file can be used for your progress report and your final report with edits, additions and comments.

Instructions for using DropBox or WebSpace have been posted. As always, if you have problems or issues, please let me know.

-Brad

Aptamer Interpretive Dance

Well, this has seemingly gone viral through the interwebs today. It is an interpretive dance about aptamer selection. I laughed my butt off, but she has done a pretty good job. I would suggest more "interactions" of potential oligonucleotides and the "target" bullseye girl. But I like that the ones that interact all do the same dance and have the same color shirts. And check out the Thriller guy. Freaking awesome.

Selection of a DNA aptamer for homocysteine using SELEX from Maureen McKeague on Vimeo.

This PhD candidate has made it to the final 4 in the AAAS "Dance Your Ph.D." competition. To view the other finalists and see all of the 2010 entries, check out: http://gonzolabs.org/dance/.

There is even one from UT: http://vimeo.com/14503172. The longhorns look positively riveted! And the guy in the video you may recognise from Andy's lab as he is one of the administrative assistants.

New better magnets

I took some hard drives apart today and usurped the magnets inside. These magnets are very powerful and pull the iron beads to the side quite quickly (full removal within 2 seconds). You can even pull the beads completely out of the liquid (good for aspirating the entire wash volume and not disturbing beads!!!) Be careful with them though, they WILL pinch fingers and it DOES hurt. Eventually, I may affix them to an 80 well rack for easier use, or you are welcome to.

-Brad

Proper care and maintenance of targets

In general, proteins should not go through freeze thaw cycles. That means the -20 is not a good place to store them prior to aliquoting if they are already resuspended. If they come dry (desicated), then they can be stored in the -20. Otherwise, please place the targets that arrive in solution in the 4C fridge till we can aliquot them (that or the next day) and store them in the -80C. For the longer term storage, we need to aliquot between 50 and 400pmol/tube depending on the concentration and number of aliquots. These tubes will get a defined box within the -80C freezer with an informational note and also the same information should be posted on the APS.

As a rule of thumb, if the product comes on dry ice, place it in the -80. If it comes on freezer packs, then place it in the -20C and if it comes chilled but not frozen or without freezer packs then it is safe to place in the 4C fridge.

So, targets that we need to take care of currently are S100B for Emma and Kamaxi and MUC-1 for Sabeena...targets should start rolling in fast now.

S100B Is IN!!!!!!!!!

Hey guys, we just got S100B and I think it's Kamaxi's. It's stored in the -80 because the -20 goes through a ton of freeze/thaw cycles which isn't idea for the protein. The info for how to resuspend/use the protein is on the whiteboard under the Aptamer Stream section.

Sterile Technique

In the past couple of weeks I have noticed that many students (not only aptamer students) are not adherent to the previously established guidelines regarding sterile technique. Here are a few reminders:

1- Don't touch your "person" with your gloves on (this includes hair, clothes, skin)
2- Don't touch your "personal belongings" with your gloves (this includes phone, notebook)

This is important for your project because there are RNases everywhere -- if they interact with your "stuff" they will affect your results.

This is also important for yourself because there are toxins everywhere -- if you touch agarose (with ethidium bromide) or acrylamide with your gloves and then grab your phone you will take these toxins with you. Your hands might not currently be touching them but they will later on (imagine a little kid touching a toilet bowl and now washing his hands, he will carry his germs with him.)

Figures for Intro/Background

In the outline for the proposal, the intro/background section says there needs to be four figures in one place, and two figures in another place. I just asked Brad, and there needs to be four figures in the intro/background. Just thought I'd clarify for anyone else that had the same question.

Aptamer Research Meeting Schedule - Tuesdays @ 4:00

9/21/10 - Lindsay Becker presents
9/28/10 - Journal Club
10/5/10 - Michael Ledbetter & Sagar Kansara present
10/12/10 - Travis Hughes & Alec Rezigh & Mayank Aranke present
10/19/10 - Emma Weiss & Katherine Li & Amanda Romero present
10/26/10 - Journal Club
11/2/10 - Jialing Fang & John Ostrominski & Ashley Dawson present
11/9/10 - Kamaxi Patel & Damilola Olatayo & Sabeena Shaikh present
11/16/10 - Stephanie Philip & Austin Rezigh & Adaobi Anyiwo present
11/23/10 - Journal Club
11/30/10 - Lab Party

Aptamer Stream Flyer

I designed a stream flyer this year for the welcome picnic.  Feel free to print it in lab, e-mail it to friends, or I can give you a full color hard copy.  The purpose of this flyer is promotional, therefore if you have friends who are interested in the FRI or are in the process of stream sorting, be sure to pass this along.  Also, for those of you who are UGTAs for Research Methods, this can be of considerable interest.

Stream Flyer 2010

Wanna selection that works?

Previous FRI students selected against Fibrinogen and Hemoglobin S (sickle cell variant). These selections seemed to be working (i.e. 27% binding to Fib & maybe 4% binding to HbS).  The selections were originally using the N71 pool on filters with standard Hepes buffer.  If you're interested in picking up where previous student left off, then let Brad know. This might be a good project to do on the side or if you just need to feel good about your bench skills.

-Gwen

Here is some of the data from the former selections.  They were actually started quite a long time ago (during the pilot year - summer 2006) by two excellent students, Fan Fan Shen and Yuxuan Wang and are well labeled.  These two students began working on other projects during the fall of 2006 and did not follow up with these.  I just haven't had the format (like this blog) to formalize a continuation project out of it.  Figure 1 is the original binding assay showing good binding to both targets.  It is actually a good figure and I hope when you post binding assay data, you label it similar to these.

Figure 1.  Binding assay to Hemoglobin S and Fibrinogen performed late summer 2006.

Sequencing on these targets was performed for R6 (Fibrinogen - Figure 2) and R6/R10 for Hemoglobin S.

Figure 2. Sequencing data trimmed down to random region

Next steps include fully analyzing the sequences, performing individual binding assays on the clones, continuing the selection more rounds and analyzing further rounds for binding and individual sequences.

Attendance Logs - please write legibly

I'm recording your lab hours for the week. It isn't always easy to read the lab attendance sheet. Please write the total hours worked (not times worked) in the little boxes on the attendance sheet & refrain from writing me notes on the sheet ... although I do love to hear from you. If there's a major screw-up on the attendance sheet, then just draw a single line through the entry & send me an email (gwenstovall@mail.utexas.edu).

Also - please continue to ask Brad, Mimi, Holly, Xinh, Nia or me to sign-off on your hours. If a mentor isn't in the lab then please put "NM" below your time.

Questions? Let me know.

Thank you,
Gwen

Meeting Signup

Next week I will hold personal meetings with all of you. We will meet in lab and then either talk in lab our outside somewhere if it's nice. You should be getting an e-mail with the doodle poll link shortly.

Puddle

As you guys can tell there is a huge puddle outside of our freezer. Great news :: this can be avoided!

Don't keep the freezer door open for an extended period of time (i.e. when you're looking through a box, just remove the box and close the door.)

Don't continuously open the door (try to get everything you need in one turn instead of having to come back for more)!

Updates

Don't forget to keep updating the site on your progress/ problems/ difficulties/ successes or over all feel of getting back into lab! I remember when I came back after 1 month of being away and it was hard to get back in! Postponing will only make it harder to fit into your routine. Also, other people can offer insight into what is going wrong. It is not a great use of your time to stall the next step when you have plenty of people that have probably had the same problem!

Trust me that you will find more than lab help because you'll notice tons of people are in your classes so you'll find class help (which is how I got through physics and organic chemistry)!

Cloning and Sequencing of 1027 selection Part 1

I ligated and cloned the sequences (hopefully). Here's how the plates looked:


The later rounds have an alphabet after them (A/B) because during the summer, I split my total reaction into two selection rounds in the hopes of finding different aptamers. Both selections are done with the same parameters. The plates were ligated, cloned, and plated with the same parameters also. All four plates show large blue colonies surrounding by smaller white colonies with larger white colonies on the periphery of the satellite colonies.

The plates were treated with X-gal and Ampicillin, the former to track which cells had the Plasmid + insert and the latter to keep cells without the plasmid from growing. They are basically “weeding out” techniques that cut down sorting time. With the plasmid + insert, the colonies should grow white on an plate with X-gal and Ampicillin, but with only the plasmid, the colonies will grow a bright blue. The smaller white colonies could be Plasmid+ insert colonies, or they could be satellite colonies which are actually blue colonies in disguise around the larger blue colony which has absorbed all of the nutrients and materials (X-gal, ampicillin, ect) from the surrounding areas. The blue colonies are blue because they have taken up a plasmid without an insert and thus the lacZ gene is not disrupted so they are able to make B-Galactoside. Since the plates were made in July, it could affect how well the ampicillin works and since the X-gal was rolled on with colli rollers, the white splatters of colonies could be the effect of uneven spreading. The white colonies are smaller, perhaps, because with the insert, the genome is now larger and takes longer to copy.



This gel was run to test whether or not the white colonies had inserts in the plasmids since they were quite small and looked like they could be satellite colonies from the large, blue colonies. The results show that although there are multiple bands for some of the colonies, there are inserts at the 300BP mark, which is where the plasmid + insert should be. The colonies were randomly chosen and rubbed with a pipette tip to gather cells for the PCR reaction. Some of them show no insert at all which could be that they grew only after the large colony had eaten all of the ampicillin.

Aliquots and -20 Freezer Reorg

We have a few 6XO dye aliquots. Whichever mentors work today will make more.

I have reorganized the -20 freezer removing boxes from old lab members and moving around the aliquots. Everything now has it's own box instead of being left in open well racks for later knocking over. In addition, I put the general volumes/concentrations for aliquots. If we are running out, then please make at least 20 tubes more. Please don't let this happen to you.

There are a few tubes unaccounted for that will be thrown out at the end of the week. PLEASE check the second shelf to see if any of these are yours. Names on the 96 well PCR racks include: Adaobi, KL/VE and Alec, but I don't know who owns the actual tubes.

-Brad

6X EtBr Required!!

6X EtBr, we are in need of it! On a separate note, I am posting this information here with a certain degree of reservation. Are notifications such as this appropriate for the blog, or should they remain confined to the dry erase board at the front of the lab?

Lab Access Limited 9/14 in the evening

From 5p-7p 9/14, Emma McInturf, David Laude's RM TA, will need to have some portion of her class in PAI 214.  The lab will be open, but she get's first priority to the front two benches.  Hope this does not affect too many people.