MeasuringE. coliand 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 totalEscherichia 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 × 107copies of the BirA target sequence present in a shake flask-derived cell sonicates sample, and for 97-9.7 × 105copies 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 × 1010copies of the T7 target sequence and matched SC qPCR over 1.54 × 104-1.54 × 107copies. 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.
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, WC1E 6BT, UK.
Alexander
A
Templar
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, WC1E 6BT, UK.
Desmond M
DM
Schofield
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, WC1E 6BT, UK.
Darren N
DN
Nesbeth
Efficiency; HCD, high cell density; LRE, linear regression of efficiency; Linear regression; OCF, optical calibration factor; PCR, Polymerase chain reaction; Pcr; SF, shake flask; Standard curve; Standardisation; Synthetic biology; WCB, working cell bank; qPCR, quantitative PCR; wcw, wet cell weight