| What's the deadline? | We would like whatever you can send by NOON FRIDAY SEPT 14. We know this is very short notice, so please send us something then, at least the bio and grant information, so that we can get it organized, and we will try to do a revision round etc. |
| Do I need to check with my Member Institution grant office? | We don't think so, since we are not nominally competing against other "internal" grants. By all means advise them, but there's not a lot of time for asking for permissions. |
| Does my inclusion mean any commitment of funds on my part? | No |
| Is the proposal limited to NYSBC affiliate faculty ? | No. We would welcome anyone from both the Member Institutions and those outside who would like to add to the proposal. Please feel free to recruit your colleagues / collaborators elsewhere. They should contact us directly at dcadmin@nysbc.org. They should register themselves on the NYSBC intranet at http://www.nysbc.org/intranetuse.htm, and consult ProteinExpressGroup. |
| 1. What is the unattended throughput (quantity and number per unit time) in terms of expression vector construction (that is with the fist stage being PCR, followed by sub cloning, transformation, colony picking, growth for miniprep, miniprep and insert verification by PCR)? | Nextgen say -- The EF has a capacity of 384 reactions of any one process at any one time. Processes can be linked where it meets with the technical needs of the customer. The times of processes are difficult to predict due to the flexibility in them with regard to the user definitions of the incubation times etc. However, an average run of PCR and clean up takes approximately 12 hours for 384 reactions. This includes a 2.5 hour PCR incubation and as we use the Promega Wizard clean up kits and the EF has one vacuum station it can process one plate at a time through the clean up. |
| What processes require human intervention? Does the robot pick colonies? | Nextgen say -- The major human intervention is at the beginning of a run with loading of the platform and at the end in clearing the platform. The processes run unattended. The EF does not pick colonies however in a number of installations we have worked with the ClonePIX?? platform from Genetix (www.genetix.com)[Quote obtained~$200k]. This system automates the spreading of cultures and the colony picking. Please note that plate incubation is a manual off line task. |
| What is the throughput in terms of expression trials (starting with verified expression vectors, transformation, growth in liquid culture, induction and harvest)? | Nextgen say -- Again this capacity is 384 reactions at once and E.coli transformation, growth, induction, harvesting, lysis, soluble/insoluble fractionation and preparation for SDS-PAGE analysis can be linked as one project. Depending on process details this can be an unattended run of approximately 40 – 48 hours for 384 samples. |
| What is the throughput in terms of protein purification (starting with over-expressing bacterial cell pellets, lysis, clarification, affinity purification, SDS-PAGE sample prep) | Nextgesn say - Again the system has a capacity of 384 samples at once. Due to the availability of one vacuum station, a process such as this could take approximately 20 – 28 hours. In affinity purification we use a batch bind process and then cast the beads into a column for washing and elution. This gives the user control of the column volume to match with the required column capacity. |
| If cloning, expression trials and purification are run in parallel, what is the throughput per month? | Nextgen -- The EF carries out one task at a time and there is no ability to run another process while, for instance, cells are growing in the incubators. A document is attached which demonstrates one approach to producing and screening 1000 constructs per month, the time it would take and approximate consumables usage (in GBP). Please note that this an example workflow only and the user has complete freedom as to how to implement the platform. |
| How many FTE's typically are required to service the robot? | Nextgen say == We suggest that one person is designated as the internal product champion and is our key contact. A user carries out virtual experiment design in OrchestratorIMS?? and then submits the project to be run on the EF. The user interface at the EF is extremely simple to use and can therefore be used by multiple users. |
| Does the robot require any non standard lab ware or consumables? | Nextgen say - A list of consumables is attached. We have tried wherever possible to use standard, widely available and best in class consumables |
| What are the built in shaking incubators specifications? | Nextgen - We have been extremely happy with the performance of these incubators and the E.coli growth performance has been compared favorably to shake flasks. Automated incubator(s) The Expressionfactory contains two separate automated incubators. The incubator is capable of being loaded automatically via a transfer station and a rear door access panel. The microtitre plates are agitated mechanically to promote cell growth to achieve an OD of greater than 3 OD units in 24-well boxes. The incubator has a front door which allows easy access for manual operations. There is an internal glass door which allows visible inspection of the content and operation of the system without disturbing the internal climate. Each incubator has two removable cassettes which house the plates used to grow cells. It is anticipated that the incubators will be loaded automatically by the robot prior to the start of a run. In this scenario, the culture plates will be loaded by the user into the plate carousels and a loading protocol will be run where the robotics will move the plates into the incubators. After this has happened the user can continue loading the robotic platform. Incubator #1 · The temperature range is 33 oC to 50 oC · There is a plate capacity for deep well plates of 14 per incubator · Temperature is controlled through the IMS software. · Plates are loaded into the incubator via a rear-door transfer station which also contains a sensor that confirms the presence of a plate on the station. This is communicated to the database software via the handling communication port. · The internal plate handler system allows the transport of plates to any storage location with full random access. · The handler consists of a vertical positioning drive, a turn drive and gripper drive. · Access time for handler is circa 20 seconds · Each incubator has: · RS 232 communications port · Transfer station connector · Option port · Each incubator has an alarm system which is activated when an abnormal status occurs or when interaction is required. · Physical dimensions are: Width 486mm Depth 412mm Height 1035mm Weight 105 kg Operating temperature range 18 – 30 oC Operating humidity 30 – 80% at 30 oC. Incubator #2 As for #1 with the exception that the incubator has a temperature control system situated on the bed of the robot that allows the temperature to be regulated down to +10 oC. |
