LabAutomation 2006 - SLAS

MP27
J. Colin Cox
Duke University Medical Center
Duke University Biochemistry
Durham, North Carolina
colin@biochem.duke.edu
Protein Fabrication Automation
LabAutomation2006
Co-Author(s)
Janel Lape
Mahmood A. Sayed
Homme W. Hellinga
Duke University Medical Center
The ability to ‘write’ a gene sequence has widespread applications in biological analysis and engineering. Rapid writing of open reading
frames (ORFs) coding for expressed proteins has the potential to transform the way by which proteins can be engineered and produced,
and has applications in protein design, synthetic biology, crystallography, etc. Here we present a process, Protein Fabrication Automation
(PFA), that facilitates the rapid de novo creation of any desired expressed ORF with low effort, high speed, and little human interaction.
The method is robust and scaleable.
Our PFA scheme is based on the total synthesis of genes from synthetic oligonucleotides and contains three main components: 1) software
to handle and manipulate ORF design (GeneFab), maintain a database of oligonucleotides and generate robotic movement scripts (FabMgr);
2) a programmable commercial liquid handling robot; 3) a genetic selection scheme to enhance yields of correctly assembled synthetic
ORFs. A wild-type protein, or scaffold, is assigned a primer assembly scheme based upon inside-out nucleation PCR gene assembly. Next,
a mutation list is provided to the PFA software package which then applies desired codon variants to a wild-type scaffold (in our applications,
these mutation lists are generated by protein design algorithms). The DNA sequences are analyzed for restriction site and codon frequency
usage, and an oligonucleotide list is generated for electronic ordering. After synthesis of the primers, FabMgr then produces a script program
for programmable liquid handling robots (here, any Tecan Genesis or Evo).
MP28
Matthew Cu
Beckman Coulter
Fullerton, California
mcu@beckman.com
Co-Author
Michael Gary Jackson
Automation of Total RNA Isolation From Cultured Eukaryotic Cells on Beckman
Coulter’s Biomek ® 3000 Laboratory Automation Workstation Using Agencourt ®
Manual isolation of total RNA can be tedious and prone to nuclease degradation due to human error. Recognizing the role of automation
in addressing these conditions, we developed an automated method on Beckman Coulter’s Biomek 3000 Laboratory Automation
Workstation to purify total RNA from cultured eukaryotic cells using Agencourt RNAPrep. Since total RNA is the starting material for a
number of downstream applications including reverse transcriptase-PCR1 (RT-PCR), quantitative real-time PCR, cDNA synthesis, cDNA
library construction and microarray analysis, confidence in the data corresponds directly to the quality of the purified RNA. Various cell lines,
in a 96-well format, were used to demonstrate the automated process for RNA isolation. Purification begins with the addition of a magnetic
lysis solution to disrupt cell membranes and bind RNA to the paramagnetic particles. RNA remains affixed to the beads during DNase
treatment and washing, and then is eluted from the particles. Purified total RNA is evaluated spectrophotometrically and then is observed
by gel electrophoresis for quality. This total RNA isolation method is the starting point for additional automated methods that conveniently
use the same deck configuration in applications such as cDNA Synthesis, In-vitro Transcription and Fragmentation.
The information presented here will include:
• The description of the automated methods
• The results obtained when using the methods.
1. The PCR process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffman-LaRoche, Ltd.
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