Article Figures & Data
Figures
- FIG 1
Tuning of plasmid copy number controls loading into vesicles. (A) Three plasmids were constructed using point mutations of the pSC101 replication origin, generating plasmids with increasing plasmid copy number, as confirmed by qPCR. (B) The number of plasmids per picogram of vesicle protein increased with increased plasmid copy number. (C) The gene transfer time decreased as plasmid copy number increased. Error bars signify standard deviations. **, P < 0.01; ***, P < 0.001.
- FIG 2
The impact of plasmid size on vesicle production and transfer. (A) Four plasmids were constructed with pLC291 origin and variable lengths of nonfunctional lambda phage DNA. Plasmid construct sizes were confirmed using restriction digestion and gel electrophoresis. (B) The number of plasmids per picogram of vesicle protein. (C) Plasmid copy number per genomic copy was quantified by qPCR. (D) Gene transfer time for vesicles containing plasmids pLC-3.5, pLC-7.5, pLC-10, and pLC-15. Error bars signify standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, P > 0.05.
- FIG 3
Impact of plasmid origin on vesicle production and size. (A) Three 3.5-kb plasmids were constructed with different origins of replication: pMB1, RK2+ColE1 (pLC), and SC101. (B) Plasmid copy number per genomic copy was quantified by qPCR. (C) Number of plasmids per picogram of vesicle protein quantified by qPCR. (D) Extracellular vesicles were isolated from E. coli cells carrying one of three plasmids types, pMB1, pLC or SC101, and used in gene transfer measurements. Error bars signify standard deviations. *, P < 0.05; ***, P < 0.001; NS, P > 0.05.
- FIG 4
Summarizing the impact of plasmid characteristics on vesicle packaging and DNA transfer rates. (A) Plasmid loading per vesicle is plotted against plasmid copy number. Lines show linear fits for each plasmid origin. (B) Gene transfer rates calculated from time-to-transfer measurements. (C) Gene transfer rates normalized to the average number of plasmids per vesicle. Error bars show standard errors. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Additional Files
Supplemental material
- Supplemental file 1 -
Tables S1 (Oligonucleotide primers) and S2 (Plasmid constructs and copy numbers); Fig. S1 (Influence of plasmid copy number on EV production), S2 and S4 (EV size distribution across plasmid copy number [S2] and plasmid sizes [S4]), S3 (Influence of plasmid size on EV production), S5 (Vesicle protein quantification from EVs loading different plasmid origins), S6 (EV size distributions produced by donor cells containing plasmids with different replication origins), S7 (Probability of data given values of r between 0 and 2/h), S8 (CT values from qPCR of EVs with and without DNase treatment), and S9 and S10 (P values for unpaired Student t test for measurements reported in Fig. 1 to 3 [S9] and 4B and C [S10]); and supplemental text
PDF, 1.1M
- Supplemental file 1 -
