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Essential validation methods for E. coli strains created by chromosome engineering.

Tiruvadi Krishnan S, Moolman MC, van Laar T, Meyer AS, Dekker NH - J Biol Eng (2015)

Bottom Line: The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape.While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays.The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, Delft, 2628 CJ The Netherlands.

ABSTRACT

Background: Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and industrial research to introduce multiple insertions in the same Escherichia coli strain. To date, λ-Red recombination (also known as recombineering) and P1 phage transduction are the most successfully implemented chromosome engineering techniques in E. coli. However, due to errors that can occur during the strain creation process, reliable validation methods are essential upon alteration of a strain's chromosome.

Results and discussion: Polymerase chain reaction (PCR)-based methods and DNA sequence analysis are rapid and powerful methods to verify successful integration of DNA sequences into a chromosome. Even though these verification methods are necessary, they may not be sufficient in detecting all errors, imposing the requirement of additional validation methods. For example, as extraneous insertions may occur during recombineering, we highlight the use of Southern blotting to detect their presence. These unwanted mutations can be removed via transducing the region of interest into the wild type chromosome using P1 phages. However, in doing so one must verify that both the P1 lysate and the strains utilized are free from contamination with temperate phages, as these can lysogenize inside a cell as a large plasmid. Thus, we illustrate various methods to probe for temperate phage contamination, including cross-streak agar and Evans Blue-Uranine (EBU) plate assays, whereby the latter is a newly reported technique for this purpose in E. coli. Lastly, we discuss methodologies for detecting defects in cell growth and shape characteristics, which should be employed as an additional check.

Conclusion: The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape. While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays. The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.

No MeSH data available.


Related in: MedlinePlus

Evaluation of E. coli strains based on cellular growth or morphology characteristics. a Growth curves of the AB1157 (black) and recombineered ΔmotAB cells (red) in shake flasks containing LB medium at 37 °C and 250 rpm. b A simple method using semi-log plot to find the log phase of the growth curve for AB1157 (black) and ΔmotAB (red) strains. The linear region of the semilog plot is the log phase of the growth curve. c The exponential fitting of the selected log phase of the growth curve for AB1157 (black) and ΔmotAB (red) strains. From the fit (dotted lines), the growth rates (μ) are determined as 1.11 h−1 and 1.13 h−1 for one sample of AB1157 and ΔmotAB strains repectively. d A sample phase contrast image of AB1157 cells which were grown in LB medium at 37 °C and 250 rpm is shown. Such images were analyzed by MicrobeTracker software to calculate precisely the cell length and volume for each cell. e & f The data of cell length and cell volume of ~350 cells for each strain are plotted using a Box and Whiskers plot. The line within the box corresponds to the median value, the borders show the upper and lower quartiles (75 % and 25 %), and the whiskers represent the maximum and minimum values
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Fig3: Evaluation of E. coli strains based on cellular growth or morphology characteristics. a Growth curves of the AB1157 (black) and recombineered ΔmotAB cells (red) in shake flasks containing LB medium at 37 °C and 250 rpm. b A simple method using semi-log plot to find the log phase of the growth curve for AB1157 (black) and ΔmotAB (red) strains. The linear region of the semilog plot is the log phase of the growth curve. c The exponential fitting of the selected log phase of the growth curve for AB1157 (black) and ΔmotAB (red) strains. From the fit (dotted lines), the growth rates (μ) are determined as 1.11 h−1 and 1.13 h−1 for one sample of AB1157 and ΔmotAB strains repectively. d A sample phase contrast image of AB1157 cells which were grown in LB medium at 37 °C and 250 rpm is shown. Such images were analyzed by MicrobeTracker software to calculate precisely the cell length and volume for each cell. e & f The data of cell length and cell volume of ~350 cells for each strain are plotted using a Box and Whiskers plot. The line within the box corresponds to the median value, the borders show the upper and lower quartiles (75 % and 25 %), and the whiskers represent the maximum and minimum values

Mentions: Bacterial growth curve analysis provides an overview of the growth behavior of the chromosomally engineered E. coli strains. A typical bacterial growth curve starts with a lag phase as the bacteria adapt to the fresh growth medium, followed by a log phase in which growth is exponential. The final phase of the growth curve displays stationary growth as a result of nutrient scarcity, after which cells eventually die (Fig. 3a) [37]. Two important parameters that can be determined using the technique of growth curve analysis are the log-phase growth rate (μ) and the duration of lag phase (τl) [38]. The log phase doubling time (generation time, τd) is calculated from μ. If growth defects are introduced during the strain creation process, they can be detected by comparing the generation times of the parental strain with those of the created strain. The literature suggests numerous models and tools with which to perform this analysis [37, 38]. As an example, we have performed growth curve validation for the AB1157 and ΔmotAB strains (Fig. 3a, Methods section IIF). The critical step is to determine which time points of the growth curve fall in the log phase; fortunately, this is easily achieved by determining the linear region of the semi-log plot of the same curve (Fig. 3b). By fitting the log phase portion of the curve with an exponential function, we calculated the growth rates for each sample (Fig.3c). From the growth rates, the mean generation times with standard deviation (SD) for the AB1157 and ΔmotAB were found to be 39.2 ± 2.1 min and 38.7 ± 1.9 min, respectively. To determine the statistical significance of this difference, we employed t-test statistics for two independent sample means [39]. From the observed p-value of 0.68 (Table 4), we conclude with 95 % confidence intervals that no significant difference in generation times can be attributed to the motAB deletion genotype.Fig. 3


Essential validation methods for E. coli strains created by chromosome engineering.

Tiruvadi Krishnan S, Moolman MC, van Laar T, Meyer AS, Dekker NH - J Biol Eng (2015)

Evaluation of E. coli strains based on cellular growth or morphology characteristics. a Growth curves of the AB1157 (black) and recombineered ΔmotAB cells (red) in shake flasks containing LB medium at 37 °C and 250 rpm. b A simple method using semi-log plot to find the log phase of the growth curve for AB1157 (black) and ΔmotAB (red) strains. The linear region of the semilog plot is the log phase of the growth curve. c The exponential fitting of the selected log phase of the growth curve for AB1157 (black) and ΔmotAB (red) strains. From the fit (dotted lines), the growth rates (μ) are determined as 1.11 h−1 and 1.13 h−1 for one sample of AB1157 and ΔmotAB strains repectively. d A sample phase contrast image of AB1157 cells which were grown in LB medium at 37 °C and 250 rpm is shown. Such images were analyzed by MicrobeTracker software to calculate precisely the cell length and volume for each cell. e & f The data of cell length and cell volume of ~350 cells for each strain are plotted using a Box and Whiskers plot. The line within the box corresponds to the median value, the borders show the upper and lower quartiles (75 % and 25 %), and the whiskers represent the maximum and minimum values
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4488041&req=5

Fig3: Evaluation of E. coli strains based on cellular growth or morphology characteristics. a Growth curves of the AB1157 (black) and recombineered ΔmotAB cells (red) in shake flasks containing LB medium at 37 °C and 250 rpm. b A simple method using semi-log plot to find the log phase of the growth curve for AB1157 (black) and ΔmotAB (red) strains. The linear region of the semilog plot is the log phase of the growth curve. c The exponential fitting of the selected log phase of the growth curve for AB1157 (black) and ΔmotAB (red) strains. From the fit (dotted lines), the growth rates (μ) are determined as 1.11 h−1 and 1.13 h−1 for one sample of AB1157 and ΔmotAB strains repectively. d A sample phase contrast image of AB1157 cells which were grown in LB medium at 37 °C and 250 rpm is shown. Such images were analyzed by MicrobeTracker software to calculate precisely the cell length and volume for each cell. e & f The data of cell length and cell volume of ~350 cells for each strain are plotted using a Box and Whiskers plot. The line within the box corresponds to the median value, the borders show the upper and lower quartiles (75 % and 25 %), and the whiskers represent the maximum and minimum values
Mentions: Bacterial growth curve analysis provides an overview of the growth behavior of the chromosomally engineered E. coli strains. A typical bacterial growth curve starts with a lag phase as the bacteria adapt to the fresh growth medium, followed by a log phase in which growth is exponential. The final phase of the growth curve displays stationary growth as a result of nutrient scarcity, after which cells eventually die (Fig. 3a) [37]. Two important parameters that can be determined using the technique of growth curve analysis are the log-phase growth rate (μ) and the duration of lag phase (τl) [38]. The log phase doubling time (generation time, τd) is calculated from μ. If growth defects are introduced during the strain creation process, they can be detected by comparing the generation times of the parental strain with those of the created strain. The literature suggests numerous models and tools with which to perform this analysis [37, 38]. As an example, we have performed growth curve validation for the AB1157 and ΔmotAB strains (Fig. 3a, Methods section IIF). The critical step is to determine which time points of the growth curve fall in the log phase; fortunately, this is easily achieved by determining the linear region of the semi-log plot of the same curve (Fig. 3b). By fitting the log phase portion of the curve with an exponential function, we calculated the growth rates for each sample (Fig.3c). From the growth rates, the mean generation times with standard deviation (SD) for the AB1157 and ΔmotAB were found to be 39.2 ± 2.1 min and 38.7 ± 1.9 min, respectively. To determine the statistical significance of this difference, we employed t-test statistics for two independent sample means [39]. From the observed p-value of 0.68 (Table 4), we conclude with 95 % confidence intervals that no significant difference in generation times can be attributed to the motAB deletion genotype.Fig. 3

Bottom Line: The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape.While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays.The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, Delft, 2628 CJ The Netherlands.

ABSTRACT

Background: Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and industrial research to introduce multiple insertions in the same Escherichia coli strain. To date, λ-Red recombination (also known as recombineering) and P1 phage transduction are the most successfully implemented chromosome engineering techniques in E. coli. However, due to errors that can occur during the strain creation process, reliable validation methods are essential upon alteration of a strain's chromosome.

Results and discussion: Polymerase chain reaction (PCR)-based methods and DNA sequence analysis are rapid and powerful methods to verify successful integration of DNA sequences into a chromosome. Even though these verification methods are necessary, they may not be sufficient in detecting all errors, imposing the requirement of additional validation methods. For example, as extraneous insertions may occur during recombineering, we highlight the use of Southern blotting to detect their presence. These unwanted mutations can be removed via transducing the region of interest into the wild type chromosome using P1 phages. However, in doing so one must verify that both the P1 lysate and the strains utilized are free from contamination with temperate phages, as these can lysogenize inside a cell as a large plasmid. Thus, we illustrate various methods to probe for temperate phage contamination, including cross-streak agar and Evans Blue-Uranine (EBU) plate assays, whereby the latter is a newly reported technique for this purpose in E. coli. Lastly, we discuss methodologies for detecting defects in cell growth and shape characteristics, which should be employed as an additional check.

Conclusion: The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape. While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays. The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.

No MeSH data available.


Related in: MedlinePlus