Analysis and Design
Information provided by the requesting lab, such as known biochemistry and planned applications, is combined with bioinformatic analysis using a variety of server-based software. This analysis evaluates predicted areas of folding and disorder, secondary structure prediction, areas of homology and biochemical activity motifs. The possible domain boundaries are indentified and potential construct limits are proposed. The gene is also analyzed for codon usage in bacteria. A cloning strategy taking advantage of a substantial vector library is developed. The resulting plan is submitted to the requesting lab for review. Clonemanager software is then used to design and analyze oligos for PCR construction that are compatible with ligation-independent cloning.
Familiarize users with server-based software for analysis including: Foldindex, DisEMBL, Globplot, Jalview and Jpred. Demonstrate how the tools are applied, how to use the information from the analysis and what to concentrate on for construct design.
Oligo design, load sequences of templates, use Clonemanager software to design, analyze and finalize sequences for ordering.
Using template provided by the requesting lab the various gene constructs are produced by PCR. PCR products are purified in 96-well plate format on the Biomek and then analyzed using the Labchip 90.
PCR fragments are prepared for LIC by treatment with T4 DNA polymerase. These are annealed into the appropriate vectors and used to transform XL1 Blue cells. Transformants are plated onto 48-well Q-trays and incubated overnight. Colonies are picked and used to inoculate 1 ml of media in each well of a 96-well block. These cultures are grown overnight and then plasmid DNA is purified by miniprepping in a 96-well plate on the Biomek. Positive clones are identified by PCR followed by Labchip analysis.
LIC protocols; guidance in our lab for PCR construction, processing for LIC, annealing, transformation and screening for positive clones, using the Biomek for minipreps and the Labchip for PCR analysis.
Positive clones obtained as for E. coli expression are used to make recombinant baculovirus using the Bac to Bac system. Recombinant bacmids are purified using traditional alkali lysis miniprep protocols. The bacmids are screened by PCR using M13 primers to confirm insertion of the gene of interest into the bacmid. The bacmid DNAs are used to transfect insect cells in suspension in 24-well blocks. Transfection in suspension has been shown to yield higher viral titers. The resulting virus is titered by QPCR. Virus titering by QPCR can be requested as an individual service.
Positive clones are identified in pCDNA3.1-based vectors that have been modified for LIC using the same cloning region found in our bacterial and baculovirus vectors. These provide a variety of fusion tags from His to FLAG and other commonly used tags.
A master plate of positive clones will be produced. The clones will be used to transform an expression strain. Overnight cultures of 1 ml will be used to produce glycerol stocks as well as inoculate 0.5 ml cultures for expression trials. After the expression incubation the cells are lysed by chemical additive and target protein is partially purified by metal affinity chromatography in 96-well plate format on the Biomek. Elutions are analyzed for the protein of interest using the Labchip 90. A spreadsheet containing the project information with the protein concentrations determined by the Labchip 90 will be given to the requesting lab. The glycerol stocks of the clones in the expression strain will be handed off along with all the protocols necessary for reproducing these results at larger scale. The master plate of positive clones will be made available if requested.
Trouble-shooting expression problems, review data and protocols, do limited bioinformatic analysis, provide HTP protocols, suggest possible experiments, help develop strategy, in some cases run limited trials and analysis.
Expression cultures are set up in deep-well, round-bottomed, 24-well plates. The MOI is set using the titering information generated by QPCR. Incubation times and other parameters are varied for optimization. Target protein is partially purified by metal affinity chromatography in 96-well plate format on the Biomek. Elutions are analyzed for the protein of interest using the Labchip 90. The data relating to expression and purification yields will be given to the requesting lab. The virus, bacmids and all the protocols necessary for reproducing these results at larger scale will be transferred to the requesting lab. The master plate of positive clones will be made available if requested.
Transient transfection will be used for expression testing. The cultures will be seeded in 5 ml volumes in 24-well blocks. This will be done in triplicate to compare expression at 24, 48 and 72- hour incubation. If necessary, longer time-points may be examined. Target protein is partially purified by metal affinity chromatography in 96-well plate format on the Biomek. Elutions are analyzed for the protein of interest using the Labchip 90. The data relating to expression and purification yields will be given to the requesting lab. The plasmid DNAs and all protocols will be transferred to the requesting lab