DNASU Plasmid Repository Using the Brooks BioStore

Transcription

1 DNASU Plasmid Repository Using the Brooks BioStore for Plasmid Storage, Distribution, and Biological Applications By Catherine Seiler Ph.D., Scientific Liaison, DNASU

2 DNASU is located in the Virginia G. Piper Center for Personalized Diagnostics at the Biodesign Institute at Arizona State University (ASU). The mission of the center is to drive the discovery of biomarkers for the early detection of diseases. One of the center s major philosophies is that it not only carries out original research, but also provides resources to the research community. The center wants to facilitate research around the world. In addition, it has other core facilities a next-generation Sanger sequencing center, a protein array facility, and a DNA purification center facility that are integrated with the lab. DNASU is a plasmid repository. Plasmids are stored in bacteria at -80 C. They are very different from clinical samples. You don t need to be as careful with them. Pre-analytical and post-analytical variables and collection and the amount of time the plasmids spend on the bench are not worrisome issues. Because plasmids are in bacteria, they can be grown. They are essentially a renewable resource. Since the why of DNASU s storage is to facilitate research around the world, the lab must be able to access the plasmids quickly and in large amounts, if needed. It s essential to be able to re-arrange the plasmids for a particular biological application in the lab, or for a customer, and to provide the correct samples. Currently, DNASU has 200,000 plasmids in its repository, and the collection is always growing. The collection has many different vectors, genes from a variety of different species, plus specialized collections. These are deposited from all over the country. DNASU has created a number of these plasmids, plus it has grant collaborations to deposit plasmids from researchers around the country. Whenever we create a plasmid, or receive one from an external researcher, we use a well-integrated set of informatics and homegrown LIMS systems, as well as automated processes, to guarantee the material s quality. All of this happens before the plasmid enters the Brooks BioStore. We have a plasmid management system where every single plasmid and plate that enters the repository to be cloned or sequenced is stored in FLEX. Everything is bar coded so it won t be lost. We do sequence validate the genes that are contained within the plasmids with our ACE sequence validation software. With this software, we can validate the insert sequence for hundreds of thousands of plasmids at one time. It s fully automated, saves a lot of time, and allows you to determine if the gene is good or bad. At this point, the plasmid data is entered into the lab s database. The physical samples are transferred into 2D bar coded tubes. Matrix tubes are used and those are entered into the BioStore. The lab also has a database that tracks protein arrays and is able to integrate things such as patient samples. Integrating the BioStore Originally, the center was located in Boston. Five years ago, when it moved to Arizona, a new storage system was evaluated for the facility. A lot of research was conducted and the lab decided to buy the Brooks BioStore. The BioStore holds 855,000 samples. It has both tube and vial pickers. While vials are not stored today, the capability was desired to store material such as blood samples in the future. The BioStore was installed in 2010 and started loading samples in January 2011 (see figure 1). Researchers who are interested in plasmids from the repository visit DNASU s Website ( and search for samples. The site is like the Amazon of plasmids. A researcher adds a plasmid to their shopping cart and checks out. At the end of the day, the lab gets a list of all of the orders placed on the system. When the list is downloaded, it includes all of the samples locations. A 2D bar code allows the technician to find the plasmid in the BioStore. The lab tracks the original plate location when the plasmid is loaded into the BioStore. The lab maintains backups in the event there are issues when 96-well plates are transferred into the BioStore. The BioStore picker selects the plasmids in the order received. The technician grows the plasmid overnight and ships the order to the user at room temperature via FedEx next-day delivery. The BioStore has significantly impacted the lab s ability to process plasmid orders. It is difficult for researchers to plan ahead. When people order things, they want them immediately. So speed is very important. The BioStore allows the lab to pick things quickly, especially very large orders that in the past may have come from 15 different plates in 15 different freezers. In 2010, it took 7 to 10 business days to fill a plasmid order. After installing the BioStore in 2011, fulfillment time dropped to 4 to 5 days. And presently, the lab is shipping in about 3 to 4 days. DNASU ships plasmids worldwide. On average, about 150 orders are filled per month. The lab has shipped over 4,500 orders for 230,000 plasmids to 47 states and 40 countries. 2

3 Using the Brooks BioStore for Plasmid Storage, Distribution, and Biological Applications BioStore Applications Speed Throughput The need to cherry pick plasmids was the main reason the BioStore was purchased. Much of the lab s work involves creating plasmid collections. The gateway cloning system is used in this process. The center is part of the ORFeome Collaboration. It has cloned about 15,000 human genes to date into a master vector. A gene insert can be transferred into any type of desired expression vector in one easy combinational cloning reaction (see figure 2). When new collections are created, the lab makes them in a master vector. If an order needs to be remade, it s picked from the BioStore. Figure 1: DNASU Brooks BioStore The DNASU team is focused on ensuring plasmid quality. All of the plasmids are sequenced before being loaded into the BioStore to make sure the gene insert is correct. Also, the plasmids are grown before they are imported into the BioStore and before distribution to a researcher. These quality-control measures ensure that the physical sample is still viable. The antibiotic resistance of the vector is also validated to ensure the correct clone. DNASU s policy is to regrow a plasmid after six freeze-thaw cycles. In reality, the lab usually runs out of samples by six freeze-thaw cycles and has to regrow them. In working with the Brooks software team we will be able to track the number of times a plasmid exits and enters the BioStore. The technology used in the lab is called NAPPA. It s a DNA programmable protein array. Instead of expressing and purifying thousands of proteins, which is a long complicated process, the DNA is spotted in a plasmid on a slide. Each spot contains a different individual gene in a plasmid backbone and has a GST tag. Once the chip is made, on-chip transcription translation can be performed and the protein captured using a GST antibody printed on each spot. The protein can be made for different genes in every single spot on the slide at the time the experiment is performed. Storage, purification, or the other things that complicate the production of protein array slides are not problems. The lab is working towards CAP biorepository certification, and it must demonstrate individual samples can be tracked in and out of the cold space. The lab has had very few problems with the BioStore. DNASU has distributed 230,000 plasmids and problems have been reported with less than 2%. Sixty percent of these were fixed, corrected, and sent back to the customer. Any plasmids found incorrect are removed from the collection. Figure 2: Plasmid creation process at DNASU. 3

4 How to Plan Next-Generation Biological Repositories If a specialized array is desired, picking the individual plasmids can be done with the BioStore. For example, the lab studies auto antibodies in the blood. There are auto antibodies that are different in patients with breast cancer than non-patients. Those antibodies in patients would bind to the individual spot that contains a specific protein and it wouldn t bind in the controls. That allows the identification of autoantibody biomarkers in the disease. The lab has automated this process to make it faster. It has an arrayer that spots the DNA on the arrays. There are two HSPro systems that do the transcription translation and screening of 48 slides at a time. The lab also has a scanner that analyzes 48 slides at a time and visualizes the antibodies bound to the spots (see figure 3). If the researcher doesn t want to express a lot of protein, and wants to spot DNA, large quantities of DNA must be produced. Imagine being asked to do 2,500 mini-preps or the 25,000 mini-preps that represent the entire human genome. The size of this task would be very frustrating. The lab encountered this problem when it wanted to make arrays containing tens of thousands of individual genes. A DNA factory was developed that can do 4,600 preps in a 96-well format in 70 hours. It s based on robots that transfer the plates between liquid handling, centrifuges, incubators, shakers, and a Brooks XPeel System. Thisprocess is integrated within DNASU and the lab can track the same bar code across its DNA purification system. The BioStore is central to DNASU s activities, but it also is facilitating global research. The center is able to provide quality materials to researchers very quickly anywhere in the world. Question: What is the main reason for any down time you might experience with the BioStore? Seiler: The BioStore s performance has been phenomenal. The main reason we have experienced little down time is the preventative maintenance program. Brooks has worked with DNASU s technicians to make sure that the maintenance happens later in the week, a time when there isn t much shipping. If there is a problem, Brooks knows about it before the lab does and fixes it immediately. But our organization really hasn t had any problems. Figure 3: Array printing and scanning. 4

5 Using the Brooks BioStore for Plasmid Storage, Distribution, and Biological Applications About the Author Dr. Cathy Seiler is the scientific liaison for the DNASU plasmid repository and the Virginia G. Piper Center for Personalized Diagnostics at the Biodesign Institute at ASU. She received her undergraduate degree in biochemistry and molecular biology from Boston University and her Ph.D. from the Watson School of Biological Sciences at Cold Spring Harbor Laboratory (CSHL). After post doctoral research in Dr. Patrick Paddison s lab at CSHL, she joined Dr. Joshua LaBaer s group at the Harvard Institute of Proteomics at Harvard Medical School as the Scientific Liaison of the PlasmID repository. In 2009, Dr. LaBaer moved to ASU along with the repository, renamed DNASU. Dr. Seiler has continued as the Scientific Liaison of DNASU handling the scientific, customer service, legal, and outreach activities for the repository, as well as leading a team of talented student scientists, programmers, and graphic designers to improve the design of and content in DNASU. She also works closely with the clinical research community in Arizona as a Governance Board member for the Arizona Biomedical Research Consortium-funded virtual biorepository, the Arizona Biospecimen Consortium. Dr. Seiler also regularly teaches undergraduate students at ASU and adult courses at the Osher Lifelong Learning Institute focusing on cancer biology and personalized medicine. You can contact Dr. Seiler at catherine.seiler@asu.edu or

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