13C surface characterization of midplane and crown collector probes on DIII-D

A dual collector probe system has been implemented on DIII-D for scrape-off-layer (SOL) impurity transport studies. These experiments injected isotopically enriched methane (13CD4) and sampled the impurities from this extrinsic, primary source with graphite collector probes at the outboard midplane and crown of upper single null L-mode plasmas. Using a stable isotopic mixing model, results suggest that 13C from methane injections prior to these experiments has built up on the walls of DIII-D to act as a secondary, intrinsic source of enriched 13C to the collector probes. This secondary source accounts for nearly 60 % of the deposits on the midplane collector probes and nearly 90 % of the deposition on the collector probes in the crown. These results lay the foundation for future impurity transport models and suggest that further simulation of impurity transport during the methane injection experiments will require two sources of enriched impurities in order to accurately model the SOL impurity profiles of 13C

pMINERVA: A donor–acceptor system for the in vivo recombineering of scFv into IgG molecules

Phage display is the most widely used method for selecting binding molecules from recombinant antibody libraries. However, validation of the phage antibodies often requires early production of the cognate full-length immunoglobulin G (IgG). The conversion of phage library outputs to a full immunoglobulin via standard subcloning is time-consuming and limits the number of clones that can be evaluated. We have developed a novel system to convert scFvs from a phage display vector directly into IgGs without any in vitro subcloning steps. This new vector system, named pMINERVA, makes clever use of site-specific bacteriophage integrases that are expressed in E. coli and intron splicing that occurs within mammalian cells. Using this system, a phage display vector contains both bacterial and mammalian regulatory regions that support antibody expression in E. coli and mammalian cells. A single-chain variable fragment (scFv) antibody is expressed on the surface of bacteriophage M13 as a genetic fusion to the gpIII coat protein. The scFv is converted to an IgG that can be expressed in mammalian cells by transducing a second E. coli strain. In that strain, the phiC31 recombinase fuses the heavy chain constant domain from an acceptor plasmid to the heavy chain variable domain and introduces controlling elements upstream of the light chain variable domain. Splicing in mammalian cells removes a synthetic intron containing the M13 gpIII gene to produce the fusion of the light chain variable domain to the constant domain. We show that phage displaying a scFv and recombinant IgGs generated using this system are expressed at wild-type levels and retain normal function. Use of the pMINERVA completely eliminates the labor-intensive subcloning and DNA sequence confirmation steps currently needed to convert a scFv into a functional IgG Ab