Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR

Abstract

BackgroundPlasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. Constant modification has brought forth countless plasmid vectors whose characteristics in terms of average plasmid copy number (PCN) and stability are rarely known. The crucial factor determining the PCN is the replication system; most replication systems in use today belong to a small number of different classes and are available through repositories like the Standard European Vector Architecture (SEVA).ResultsIn this study, the PCN was determined in a set of seven SEVA-based expression plasmids only differing in the replication system. The average PCN for all constructs was determined by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, respectively.ConclusionsThe average PCN determined here for a set of standardized plasmids was generally at the lower end of previously reported ranges and not related to the degree of heterogeneity. Further characterization of a heterogeneous and a homogeneous population demonstrated considerable differences in the PCN of sub-populations. We therefore present direct molecular evidence that the average PCN does not represent the true number of plasmid molecules in individual cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0610-8) contains supplementary material, which is available to authorized users.

Improved Thermotolerance of Genome‐Reduced Pseudomonas putida EM42 Enables Effective Functioning of the PL/cI857 System

“…The c I857/P L heat‐inducible system of lambda phage was reconstructed in vectors pSEVA2214 (RK2 origin of replication, low plasmid copy number PCN), pSEVA2314 (pBBR1 origin, medium PCN), and pSEVA2514 (RSF1010 origin, high PCN), the sequences of which can be found in GenBank under accession numbers MH650998, MH650997, and MH638301, respectively. Their PCNs for E. coli were accurately determined by Jahn et al with a droplet digital PCR method . Note however that RSF1010 replicon borne by the SEVA plasmid pSEVA2514 utilized in this work is not identical to the wild‐type origin of replication, but has a stronger promoter in front of the replication proteins that increases copy number by 3‐fold .…”

“…Their PCNs for E. coli were accurately determined by Jahn et al with a droplet digital PCR method. [26] Note however that RSF1010 replicon borne by the SEVA plasmid pSEVA2514 utilized in this work is not identical to the wild-type origin of replication, but has a stronger promoter in front of the replication proteins that increases copy number by 3-fold. [27] Regardless of specific copy numbers, the hierarchy of relative plasmid abundance in P. putida of the vectors used is RS1010 (high) > pBBR1 (medium) > RK2 (low).…”

Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology

“…Surprisingly, expression from the medium-copy #6 was lower than expected. Jahn et al (33) measured the copy number of different SEVA vectors and found oriV #6 to have a higher copy number than #7, although differences in expression and copy number could be due to a different strain being used. end of the part ( Figure 1B), with CT terminators including C-terminus stop codon.…”

Restoration of wild-type motility to flagellin-knockoutEscherichia coli

“…Each of the starting plasmids contained a copy of fliC from E. coli AW405 under control of either its natural promoter (pJ211) or the IPTG-inducible T5 promoter (pJ291 and pD441), and with either the medium copy-number p15a replication origin (med) or the high copy-number pUC origin (hi). To most closely replicate the low gene dosage of chromosome-encoded genes, we also selected the RK2 replication origin, which has previously been shown to consistently maintain plasmids with low copy numbers of ~5 per cell [13,14]. The two promoters on the set of starting plasmids provided for either endogenously-controlled or inducible expression.…”