A synthetic biology standard for Chinese Hamster Ovary cell genome monitoring and contaminant detection by polymerase chain reaction.

BACKGROUND: Chinese Hamster Ovary (CHO) cells are the current industry standard for production of therapeutic monoclonal antibodies at commercial scales. Production optimisation in CHO cells hinges on analytical technologies such as the use of the polymerase chain reaction (PCR) to quantify genetic factors within the CHO genome and to detect the presence of contaminant organisms. PCR-based assays, whilst sensitive and accurate, are limited by (i) requiring lengthy sample preparation and (ii) a lack of standardisation. RESULTS: In this study we directly assess for the first time the effect of CHO cellular material on quantitative PCR (qPCR) and end-point PCR (e-pPCR) when used to measure and detect copies of a CHO genomic locus and a mycoplasma sequence. We also perform the first head-to-head comparison of the performance of a conventional qPCR method to that of the novel linear regression of efficiency (LRE) method when used to perform absolute qPCR on CHO-derived material. LRE qPCR features the putatively universal 'CAL1' standard. CONCLUSIONS: We find that sample preparation is required for accurate quantitation of a genomic target locus, but mycoplasma DNA sequences can be detected in the presence of high concentrations of CHO cellular material. The LRE qPCR method matches performance of a conventional qPCR approach and as such we invite the synthetic biology community to adopt CAL1 as a synthetic biology calibration standard for qPCR

Promoter engineering to optimise recombinant periplasmic Fab' fragment production in Escherichia coli

Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to the improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterised the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity

The Identification and Quantification of Sewage Contamination in the Milwaukee Estuary

Sewage contamination from failing infrastructure and sewer overflows is a major environmental and human health concern in waterways, especially in urban communities bordering the Great Lakes such as Milwaukee, Wisconsin. Culture-based fecal indicator bacteria, such as Escherichia coli, enterococci, and fecal coliforms are traditionally used to indicate the presence of a human health risk due to fecal contamination. These indicators, however, fail to distinguish between sources of fecal contamination (human vs. non-human). Two human-specific fecal indicators, human Bacteroides and human Lachnospiraceae, were used to identify and quantify sewage contamination in the Milwaukee estuary, which discharges to Lake Michigan, as well as the Milwaukee, Menomonee, and Kinnickinnic Rivers immediately upstream. Chapter 1 provides an overview of the health and environmental impacts of fecal pollution in waterways and the use of alternative indicators to track sewage pollution. Chapter 2 describes the concentrations of human fecal indicators, used as a proxy for human sewage, in the three urban rivers upstream of the Milwaukee estuary and how this information can be used for the implementation stage of the current fecal coliforms Total Maximum Daily Load (TMDL) process. Chapter 3 describes how human fecal indicators were used to characterize sewage contamination across the hydrograph. Intensive monitoring at sites in the rivers and the estuary was used to calculate event loads for storm and combined sewer overflow events and investigate relationships between loads and the degree of watershed urbanization and the amount of rainfall during an event. Chapter 4 discusses how the information generated in this research can be used in the TMDL implementation process and can be used to focus efforts of local agencies and municipalities to investigate and remediate unrecognized sources of sewage contamination. More specific information about sources of fecal pollution will be useful to create appropriate water quality goals to address the human health concerns of sewage contamination

Industrially Robust Synthetic Biology Standards for the Polymerase Chain Reaction

Synthetic Biology is ushering in a new era where reengineered genomes can enhance the capacity of host cells to produce biologic and chemical products. Standardisation is a key component of synthetic biology as it enables effective implementation (Müller and Arndt, 2012). This project has successfully generated synthetic biology standards for the quantitative polymerase chain reaction (qPCR), a highly specific and sensitive analytical platform, in order to increase its robustness for monitoring of host cell processes in an industrial setting. This project has also increased the assay throughput to allow for at-line analysis, in accordance with initiatives such as Process Analytical Technology (PAT) (Gnoth et al., 2007). Analysis was conducted on three commonly used host cell chassis and industrial contamination was also simulated by the addition of plasmid proxies. All assays were optimised by primer design and screening to ensure accuracy. End point PCR (e –pPCR) and quantitative PCR (qPCR) was conducted in the presence and absence of cellular material disrupted by a mild sonication procedure. We found that, whilst cellular material reduces assay sensitivity for a genomic locus, the presence of contaminating species can be accurately quantified. We also employed LRE–qPCR, which uses the CAL1 standard for quantification. LRE-qPCR matched the accuracy of a conventional standard curve qPCR method and we propose it as a Synthetic Biology standard. We next developed a modified standard curve method that streamlined methodology and bypassed errors inherent to the gold standard methodology to, for the first time, enable quantification of multiple targets from a single standard curve. The CyCal curve is a standard curve constructed from the CAL1 standard combined with the Cy0 data analysis. The approach was validated against 6 bioprocess targets and it was found that CyCal was able to replicate the accuracy of the gold standard approach. We then used CyCal to accurately determine how host cell plasmid copy number (PCN) evolves during fermentation. The combination of rapid sample preparation and a universal standard means that CyCal is capable of becoming the basis of an at-line qPCR assay when conducted on modern ultra-rapid qPCR thermocycler technology

Transforming uncertainty from a negative to a positive: assessing a novel intervention designed to increase uncertainty tolerance in individuals and teams

This paper, presented at the CIPD’s Applied Research Conference, uses a novel approach to explore whether tolerance to uncertainty in the workplace can be improved and therefore affect employee’s resilience, wellbeing, collaboration and sense of psychological safety

Measuring E. coli and bacteriophage DNA in cell sonicates to evaluate the CAL1 reaction as a synthetic biology standard for qPCR

We measured the impact of the presence of total Escherichia coli (E. coli) cellular material on the performance of the Linear Regression of Efficiency (LRE) method of absolute quantitative PCR (LRE qPCR), which features the putatively universal CAL1 calibration reaction, which we propose as a synthetic biology standard. We firstly used a qPCR reaction in which a sequence present in the lone genomic BirA locus is amplified. Amplification efficiency for this reaction, a key metric for many quantitative qPCR methods, was inhibited by cellular material from bioreactor cultivation to a greater extent than material from shake flask cultivation. We then compared LRE qPCR to the Standard Curve method of absolute qPCR (SC qPCR). LRE qPCR method matched the performance of the SC qPCR when used to measure 417–4.17 × 107 copies of the BirA target sequence present in a shake flask-derived cell sonicates sample, and for 97–9.7 × 105 copies in the equivalent bioreactor-derived sample. A plasmid-encoded T7 bacteriophage sequence was next used to compare the methods. In the presence of cell sonicates from samples of up to OD600 = 160, LRE qPCR outperformed SC qPCR in the range of 1.54 × 108–1.54 × 1010 copies of the T7 target sequence and matched SC qPCR over 1.54 × 104–1.54 × 107 copies. These data suggest the CAL1 standard, combined with the LRE qPCR method, represents an attractive choice as a synthetic biology qPCR standard that performs well even when unpurified industrial samples are used as the source of template material

Evaluation of enteral formulas for nutrition, health, and quality of life among stroke patients

Enteral nutritional support has been used via tube feeding for dysphagic stroke patients. We performed long and short term trials to evaluate the effects of commercial enteral nutritional supports on nutrition and health in stroke patients (mRS = 3~5) and quality of life in their caregivers. For a long term study, we recruited chronic (≥ 1 yrs) stroke patients (n = 6) and administered them 6 cans/day (1,200 kcal) of the commercial enteral formula N for 6 months according to IRB-approved protocol. We collected peripheral blood at 0, 2, 4 and 6 months. For a short term study, we recruited acute (≤ 3 months) stroke patients (n = 12) and randomly administered them two different commercial enteral formulas, N or J, for 2 weeks. We collected their blood at 0, 4, 7 and 14 day of the administration. Blood samples were analyzed to quantify 19 health and nutritional biomarkers and an oxidative stress biomarker, malondialdehyde (MDA). In order to evaluate quality of life, we also obtained the sense of competence questionnaire (SCQ) from all caregivers at 'before' and 'after trials'. As results, the enteral formula, N, improved hemoglobin and hematocrit levels in the long term trial and maintained most of biomarkers within normal ranges. The SCQ levels of caregivers were improved in the long term treatment (P < 0.05). In a case of the short term study, both of enteral formulas were helpful to maintain nutritional status of the patients. In addition, MDA levels were decreased in the acute patients following formula consumption (0.05 < P < 0.1). Most of health and nutrition outcomes were not different, even though there is a big difference in price of the two products. Thus, we evaluate the formula N has equal nutritional efficacy compared to the formula J. In addition, long term use of enteral formula N can be useful to health and nutrition of stroke patients, and the quality of life for their caregivers

Serum prolidase activity, oxidative stress, and nitric oxide levels in patients with bladder cancer

Prolidase is a member of the matrix metalloproteinase family. It plays a major role in collagen turnover, matrix remodeling and cell growth. Nitric oxide (NO) regulates many processes such as collagen synthesis and matrix remodeling. Thus, NO may augment angiogenesis, tumor invasion, and metastasis. The aim of this study was to investigate total antioxidant status (TAS), malondialdehyde (MDA) and NO levels in patients with bladder cancer and to determine their relationship with prolidase activity