Flow cytometry-based viability staining: an at-line tool for bioprocess monitoring of Sulfolobus acidocaldarius.

Determination of the viability, ratio of dead and live cell populations, of Sulfolobus acidocaldarius is still being done by tedious and material-intensive plating assays that can only provide time-lagged results. Although S. acidocaldarius, an extremophilic Archaeon thriving at 75 °C and pH 3.0, and related species harbor great potential for the exploitation as production hosts and biocatalysts in biotechnological applications, no industrial processes have been established yet. One hindrance is that during development and scaling of industrial bioprocesses timely monitoring of the impact of process parameters on the cultivated organism is crucial-a task that cannot be fulfilled by traditional plating assays. As alternative, flow cytometry (FCM) promises a fast and reliable method for viability assessment via the use of fluorescent dyes. In this study, commercially available fluorescent dyes applicable in S. acidocaldarius were identified. The dyes, fluorescein diacetate and concanavalin A conjugated with rhodamine, were discovered to be suitable for viability determination via FCM. For showing the applicability of the developed at-line tool for bioprocess monitoring, a chemostat cultivation on a defined growth medium at 75 °C, pH 3.0 was conducted. Over the timeframe of 800 h, this developed FCM method was compared to the plating assay by monitoring the change in viability upon controlled pH shifts. Both methods detected an impact on the viability at pH values of 2.0 and 1.5 when compared to pH 3.0. A logarithmic relationship between the viability observed via plating assay and via FCM was observed.

Fragmentation of Pooled PCR Products for Highly Multiplexed TILLING.

Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multi-dimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for TILLING highly pooled samples. Sequencing performance was evaluated in a total of 32 pooled PCR products produced from 4096 chemically mutagenized Hordeum vulgare DNAs pooled in three dimensions. Evaluation of read coverage and base quality across amplicons suggests this approach is suitable for high-throughput TILLING and other applications employing highly pooled complex sampling schemes. Induced mutations previously identified in a traditional TILLING screen were recovered in this dataset further supporting the efficacy of the approach.

Deletion analysis of the 3' long terminal repeat sequence of plant retrotransposon Tto1 identifies 125 base pairs redundancy as sufficient for first strand transfer.

Retroviruses and many retrotransposons are flanked by sequence repeats called long terminal repeats (LTRs). These sequences contain a promoter region, which is active in the 5' LTR, and transcription termination signals, which are active in the LTR copy present at the 3' end. A section in the middle of the LTR, called Redundancy region, occurs at both ends of the mRNA. Here we show that in the copia type retrotransposon Tto1, the promoter and terminator functions of the LTR can be supplied by heterologous sequences, thereby converting the LTR into a significantly shorter sub-terminal repeat. An engineered Tto1 element with 125 instead of the usual 574 base pairs repeated in the 5' and 3' region can still promote strand transfer during cDNA synthesis, defining a minimal Redundancy region for this element. Based on this finding, we propose a model for first strand transfer of Tto1.

A synthetic biology approach allows inducible retrotransposition in whole plants.

UNLABELLED: Retrotransposons are mobile genetic elements that transpose by reverse transcription of element RNA, followed by insertion of the cDNA into new positions of the host genome. Although they are major constituents of eukaryotic genomes, many facets of their biology remain to be understood. Transposition is generally rare, suggesting that it is subject to tight regulation. However, only the first regulatory step (transcriptional induction) is currently amenable to investigation in higher eukaryotes. To investigate the complete life cycle of a long terminal repeat (LTR) retrotransposon in plants, we established a synthetic biology program on tobacco retrotransposon Tto1, and achieved transposition in whole plants triggered by an inducible promoter. The engineered element, iTto (inducible Tto1), is a novel tool for analysis of retrotransposition in plants. In addition, it allows to explore the potential of an inducible retrotransposon for insertional mutagenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-010-9053-4) contains supplementary material, which is available to authorized users.

Virus-like particle formation and translational start site choice of the plant retrotransposon Tto1.

Ty1/copia group retrotransposon Tto1 from tobacco was put under control of an inducible promoter for expression in Arabidopsis thaliana. The system was used to analyze intermediates of the transposition process. The Tto1 RNA 5' region has a complex structure and contains several AUG codons. We therefore sought to experimentally define the translation initiation site. Constructs starting at various positions within the structural gag region were expressed in planta and functionally characterized. We found that gag proteins starting at the first ATG of the gag-pol ORF (ATG1), but also those starting at the third ATG of the gag-pol ORF (ATG3), can form virus-like particles (VLPs). However, gag protein expressed by the inducible Tto1 element had a size similar to gag starting at ATG1, and mutation of ATG1 in the inducible element abolished reverse transcription. This suggested that translation initiation at ATG1 is essential for the Tto1 life cycle. To support this conjecture, gag protein starting at ATG1, or gag protein shortened amino-terminally by nine amino acids (starting at the second ATG of the gag region, ATG2), was co-expressed with Tto1 carrying mutations at ATG1 and ATG2. Trans-complementation of the defective Tto element by gag starting at ATG1, but not by gag starting at ATG2, defines ATG1 as the functional translation initiation site.