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

Abstract

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.