Escherichia coli grown in inexpensive conical flat-bottom polypropylene tubes produce a high level of pUC vector

Abstract

The pUC-derived plasmid yield from E. coli using polypropylene tubes (PP) was compared among round and conical tubes. The yield from cells grown in a cheaper conical-PP with flat-bottom was 1.5-fold higher (p<0.001) than other PP. The use of the conical-PP can save research budgets in the current inflationary environment with nearly 5-fold higher cost efficiency.

Our laboratory has investigated the mechanism of drug resistance and virus fitness of Human Immunodeficiency Virus type-1 (HIV-1) mutants in the blood of people living with HIV-1 infection (PLWH) (Imamichi et al., 2021). Prior to the onset of the coronavirus disease 2019 (COVID-19) pandemic, we cultivated Escherichia coli, TOP10 strain (Invitrogen, Thermo Fisher Science, Waltham, MA, USA) transformed with pCR2.1 (Invitrogen) containing a partial length of HIV proviral DNA (pCR.NL.A/E, total plasmid size: 7.5-kbp) (Imamichi et al., 2021), or E.coli Stbl3 strain (Invitrogen) containing pNL4.3PB, a pUC18-derived plasmid containing a modified full-length HIV proviral DNA (total plasmid size: 15-kbp) (Imamichi et al., 2000), in Falcon-round-bottom-polypropylene (PP) tubes (F-Round-PP, Cat# 352098, Falcon, Becton Dickinson, Franklin Lakes, NJ, USA, a list price for 500 tubes: US$ 425) (Figure 1) and isolated the plasmid using the S.N.A.P. Plasmid DNA MiniPrep Kit (Invitrogen). PP tubes were optimal because they have a high tensile strength and impact resistance and exhibit good resistance to stress cracking (Weidenfeller et al., 2004). In contrast, polystyrene (PS) is relatively brittle, has lower impact resistance than PP, and is more prone to stress cracking under high-speed centrifugation (Halimatudahliana and Nasir, 2002). Therefore, we have used the PP tubes to avoid cracking during centrifugation even though it has lower thermal conductivity than PS (Han and Fina, 2011; Weidenfeller et al., 2004). Obtaining sufficient quantities of F-Round-PP became difficult during the pandemic owing to a shortage of materials and an increase in the price of these materials and different reagents compounded by restrictions in research budgets. Consequently, the cells were grown in any other available tubes. Notably, we observed that the plasmid yield from cells grown in Oxford-conical-polypropylene tubes (O-Conical-PP, Cat #OCT-15B, Oxford Lab products, San Diego, CA, USA, a list price for 500 tubes: US$135) (Figure 1) was higher than that from F-Round-PP, although the price of O-Conical-PP was only one-third of F-Round-PP. Therefore, we considered that some inexpensive PP tubes might be beneficial for laboratory budgetary considerations.

Figure 1.

Comparison of the structure of each tube.

Images of caps and bottoms of structures (A) external views of caps, (B) internal views of caps, (C) top views of the five tubes, and (D) side views with bottom shapes of the five tubes: F-Round PP, O-Conical PP, FB-Conical PP, C-Conical PP and F-Conical PS.

To evaluate the effect of PP tubes on plasmid yield, the yield was compared among O-conical-PP, Fisher-Brand-conical-PP tubes (FB-conical PP, Cat# 05–539-5, Fisher, Waltham, MA, USA, a list price for 500 tubes: US$353), and Corning conical PP tubes (C-Conical-PP, Cat # 430052, Corning, Corning, NY, USA, a list price for 500 tubes: US$480). We also used Falcon-conical-polystyrene (PS) tubes (F-Conical-PS, Cat# 352095, a list price for 500 tubes: US$414) as a control for cell growth. As shown in Figure 1, the cap sealing system of each tube is unique: The F-Round-PP has a snap-cap; the O-Conical-PP features a flat top. In contrast, the FB-Conical-PP employs a double-threaded cap system; the C-Conical-PP utilizes a plug seal cap system, and the F-Conical-PS has a dome-seal double-threaded cap system. Since oxygen transfer rate significantly affects plasmid DNA yield (Lara et al., 2019; Listner et al., 2006), it was hypothesized that the airflow in the tube could affect the difference in DNA yield; therefore, during the incubation period, the caps were placed on the tubes without sealing and taped to the tubes to avoid evaporation. Cells were cultured in 1/5 volume of each tube capacity for 12 hours at 37°C with an orbital speed at 225 rpm. All tubes were tilted 30 degrees from the vertical during incubation.

The cell growth (n=5 per each tube) was measured by optical density (OD) at 600 nm wavelength (OD600) using a SpectraMax M5 (Molecular Devices, Silicon Valley, CA, USA). The results demonstrated that the values in OD600 for E.coli growing in the O-Conical PP was slightly higher than all of the others. The determination of bacterial growth by OD600 is not an absolute value (Beal et al., 2020; Stevenson et al., 2016). Therefore, the nonparametric Friedman test (https://www.statskingdom.com/index.html) was used to statistically compare the differences. The results revealed that they were not significantly different among all tested samples containing pCR2.1 vector (Figure 2A), and the A260/A280 ratio for the purified plasmid was similar among all samples (1.89 ~1.90) (n=15). However, and of interest, the one-way ANOVA test illustrated that the normalized DNA yield (plasmid DNA yield/OD600) from O-Conical-PP was increased nearly1.5-fold significantly higher than that from other PP (p<0.001) and the yield was similar to that of F-Conical-PS (Figure 2B). To determine whether the increased output effect was E. coli strain- or plasmid size-dependent, we compared the similar parameters by extraction of pNL4.3PB (15-kbp) from the Stbl3 strain. As shown in Figure 2C, the Friedman test showed that the cell growth indicated by OD600 was not significantly different among the five tubes, but one-way ANOVA test illustrated that the normalized DNA yield was significantly increased by 1.5-fold (n=5) in O-Conical-PP compared to other brands of conical-PP tubes. In contrast, the amounts of plasmid DNA from F-Round-PP, O-Conical-PP and-Conical-PS were comparable (Figure 2D). As illustrated in Figure 1A, the cap of the FB-Conical-PP and C-Conical-PP have a plug seal system, and E.coli in tubes with the seal system exhibited reduced the plasmid yield, although this has no significant impact on cell growth. It was assumed that the cap system might inhibit aeration even when the tubes were not sealed. To elucidate the cap effect, TOP-10 strains containing 7.5-kbp plasmid were cultured in the O-Conical-PP tubes with different caps and the cell growth and DNA yield were analyzed. The results demonstrated no change in either cell growth or DNA yield (p>0.05, n=3; Supplemental Figure S1). These data consistently indicated that we obtained a significantly greater quantity of plasmid from E. coli grown in less expensive O-Conical-PP tubes than other PP tubes, regardless of the E. coli strain, plasmid size or cap sealing systems.

Figure 2.

Comparison of E. coli growth and plasmid DNA yields among different tubes.

TOP10 strain containing 7.5-kbp pCR2.1 vector (A and B) or Stbl3 strain containing 15-kbp pUC18-derived vector (C and D) were cultured in five tubes. (A and C) Cell growth and (B and D) Normalized DNA amounts. The results indicate means ±SE from five independent assay (n=5). n.s.: not significant, *: p<0.05, **: p<0.01, ***: p<0.001.

At present, the precise manufacturing process of each tube is currently unknown, and tube specifications of some, but not all tube are unavailable from manufactures. To ascertain the thickness of the wall of each tube, a direct measurement of outer diameter and inner diameter of each tube type was taken using three tubes for each, using a caliper gauge (Mitutoyo USA, Aurora, IL, USA). After measurement of the size, the wall thickness of each tube was calculated (Supplemental Table 1). The results of the ANOVA analysis indicated that there was no significant difference in the thickness among O-Conical-PP, FB-Conical-PP and C-Conical-PP tubes (Supplemental Figure S2). Therefore, it can be postulated that the wall thickness does not influence the increase in DNA yield in O-Conical-PP tubes.

The O-Conical-PP tubes have a flat-tipped shape at the bottom, whereas the other PP tubes have pointed ends (Figure 1C). Convection and thermal conductivity of the liquid are affected by the shape of the bottom of the vessel. This is demonstrated by the use of U-shaped, V-shaped, and flat bottoms in 96-well plates or flasks (Herva, 1977; Shafaie et al., 2017), Therefore, the difference at the bottom of O-Conical-PP may affect the liquid convection, the thermal conductivity of the liquid, and oxygen diffusion, which resulted in the increased plasmid DNA yield. The thermal conductivity of PS is greater than that of PP (Han and Fina, 2011; Weidenfeller et al., 2004), which has a significant effect on the rate of heating of the culture medium in PS, particularly on the initiation of cell growth. Given the observed tendency for cell growth in the O-Conical PP tubes was merely greater than that in other Conical-PP tubes and similar to that observed in F-Conical-PS tube, it was postulated that O-Conical PP tubes containing unknown components may induce a higher thermal conductivity than other PP tubes (Patti and Acierno, 2020), subsequently resulting in a higher yield of plasmid DNA than other tubes. To define the molecular mechanism behind this difference in DNA yield, it is necessary to precisely compare the thermal properties of different plastic types and oxygen diffusion.

In the current report, given that both tested plasmids are derived from the pUC ori (a high copy number) (Vieira and Messing, 1982), to ascertain the generalizability of our findings in other ori, it is necessary to compare different plasmids (Couturier et al., 1988; Shafferman and Helinski, 1983). To assess whether the increase in plasmid yield was caused only by high copy plasmids, we performed a comparative study using pBR322 (4.4-kbp, ThermoFisher), a low-to-medium-copy plasmid, in the TOP10 strain. Although statistical analysis showed no significant difference among tubes in the cell growth and DNA yield, we observed a similar trend with O-Conical-PP, O-Conical-PP and F-Conical-PS tubes. The cell growth in both tubes produced a higher yield of plasmid DNA (Supplementary Figure S3B). Our findings indicated that the use of the low-cost tube, which reduces laboratory expenses, results in the acquisition of greater quantities of plasmid DNA for downstream analysis. Further studies are necessary to determine the generalizability of our findings using not only different plasmids but also with other E. coli strains and other brands of tubes with varying tube shapes at the bottom.

The list price of O-Conical PP tubes is approximately one-third of other PP tubes, yet plasmid yield was significantly increased nearly 1.5-fold. Therefore, a cost performance index (DNA yield per tube price) of O-Conical-PP tube was 3 to 6-times higher than that of other tubes (Table 1). It is recommended that researchers who employ recombinant DNA methodologies in molecular biology reassess their laboratory supplies to identify more cost-effective options for obtaining plasmid DNA.

Table 1.

Comparison of Cost Performance Index (CPI)¹

	Tube Types
	F-Round-PP	O-Conical-PP	FB-Conical-PP	C-Conical-PP	F-Conical-PS
Price for 500 tubes2	425	135	353	480	414
Price $ per tube	0.850	0.270	0.706	0.960	0.828
7.5 Kbp DNA yield per tube 2	127	175	110	118	175
CPI for 7.5 Kbp plasmid3	149	648	156	123	211
Fold difference (×10 ^−1) 4	4.3	1.0	4.2	5.3	3.1
15 Kbp DNA yield per tube2	135	157	99	100	140
CPI for 15 Kbp plasmid 3	159	581	140	104	169
Fold difference (×10 ^−1) 4	3.7	1.0	4.1	5.6	3.4

^1:The listed price for 500 tubes of USA$ as of March 2024.

^2:The DNA yield of average from five independent assay for each tube type.

^3:The value of DNA yield per tube was divided by price$ per tube

^4:Each CPI was compared to the CPI of O-Conical-PP

Highlights.

• The cost of laboratory supplies increased due to the COVID-19 pandemic.

• The pUC DNA yields from E. coli grown in different brands of tubes were compared.

• The use of conical flat-bottom polypropylene tubes increased the DNA yield by 50%.

• The price of the tubes was one-third that of other tubes.

• A proposal to reassess lab supplies for identifying more cost-effective options is presented.