Part:BBa_K4721003
Ptuf
This part has been described by Schada Von Borzyskowski et al. (2015) [1]. The source of this part is the putative promoter region of EF-Tu, a translation elongation factor thermo-unstable in M. extorquens AM1.
Figure 1: Schematic overview of the mCherry reporter under the control of the Ptuf promoter
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Parts Collection 'Promoters for M. extorquens'
The iGEM Leiden 2023 team categorized and characterized promoters for controlled gene expression, suitable for genetic engineering of Methylobacterium extorquens AM1. This part belongs to a part collection of promoters.
Our parts collection contains multiple inducible and constitutively active promoters. We tested four constitutive promoters: PmxaF, PfumC, PcoxB and Ptuf, and two inducible promoters: IPTG inducible promoter PL/O4/A1 and vanillate inducible promoter PV10. The promoters are characterized on their respective pages.
Table 1: Overview of the constitutive promoters in the parts collection
| Name | Part | Promoter strength |
| PfumC | BBa_K4721000 | Low |
| PcoxB | BBa_K4721001 | Low |
| PmxaF | BBa_K4721002 | High |
| Ptuf | BBa_K4721003 | Medium |
Table 2: Overview of the composite inducible promoters in the parts collection
| Name Composite Part | Composite Part | Name Basic Parts | Basic Parts | Basic Part Features |
| PL/O4/A1 IPTG inducible system | BBa_K4721006 | PL/O4/A1 | BBa_K4721004 | Promoter, Operator |
| lacIq promoter | BBa_K4721005 | Promoter | ||
| lacIq gene | BBa_K1222003 | Coding | ||
| lacIq terminator | BBa_K4721007 | Terminator | ||
| PV10 vanillate inducible system | BBa_K4721008 | VanR repressor gene | BBa_K4721009 | Coding |
| RBS to combine with Pbla-mut1T promoter | BBa_K4721013 | RBS | ||
| Pbla-mut1T | BBa_K4721012 | Promoter | ||
| PV10 | BBa_K4721011 | RBS, Operator | ||
| RBS to combine with PV10 | BBa_K4721010 | RBS |
Results
Overview Promoter strengh Parts Collection
In order to characterize each promoter, each promoter was cloned in the pTE100 empty vector (Addgene 59395) (Schada von Borzyskowski et al., 2015)[1] containing an oriV-traJ’ origin with TcR. An mCherry testing cassette containing an RBS was placed under the control of the respective promoter. Each plasmid was transformed into M. extorquens AM1.
Promoter activity was tracked by measuring the mCherry fluorescence. Please read below for the detailed experimental set-up. Figure 2 shows an overview of the promoter activity for the different parts in this collection. Of the constitutive promoters, PmxaF shows the highest, followed by Ptuf. PfumC and PcoxB showed similar relatively low expressions. The IPTG inducible promoter PL/O4/A1 and the vanillate inducible promoter PV10 show significantly increased fluorescence when induced, where the induction of the IPTG inducible promoter PL/O4/A1 is the strongest.
Figure 2: Normalized expression of four constitutive promoters, and of two inducible promoters both in uninduced (-) and induced conditions (+). The bar shows the mean of the biological and technical duplicates. The error bars represent the standard deviation. The dotted line serves as a visual aid and represents the fluorescence signal of 250 nM of the Sulforhomadine from the iGEM calibration kit, as part of the Interlab study. For PL/O4/A1, a 190% increase in expression was seen when induced compared to non induced condition (Student t-test, p<0.0001). A 30% increased expression was seen for PV10 when grown under induced conditions (Student t-test, p<0.05).
Methods
In order to characterize the promoter strength, a growth experiment was set up. As a control, a strain carrying a plasmid containing the mCherry testing cassette, without a promoter, was used. All strains were inoculated at an OD600nm of 0.05. Each well contained 200 µL of standard Minimal Methanol Medium [2] used for M. extorquens. To minimize the concentration of the inducer vehicle, a 1000x stock was added to the sample to yield a 1x final concentration. The set-up of the experiment was with biological and technical duplicates. The strains were grown for five days in the Tecan Plate reader Infinite 200 PRO with a clear flat bottom black 96-well plate. Absorbance and fluorescence were measured every ten minutes, between which the plate was shaken with an linear amplitude of 1 mm. Incubation temperature was 30 ºC. Absorbance was measured at 600 nm with 25 flashes and a bandwidth of 9 nm and a settle time of 0 ms. Fluorescence was measured using fluorescence top reading using 25 flashes with an excitation wavelength of 575 nm (bandwidth 9 nm) and emission wavelength of 610 nm (bandwidth 20 nm). For this the gain was 100. An integration time of 20 µs was used and a lag and settle time of 0 µs. Z-position was 20,000 µm. After measurement of the absorbance, the real OD600nm was calculated using the M. extorquens specific formula: OD600nm = (absorption in well - 0.0755)/0.2344. The fluorescence, OD600nm and fluorescence/OD600nm are compared for each promoter as a measure for promoter strength.
Constitutive promoters
From here on, we will further discuss the results for the constitutive promoters. We have compared Ptuf relative to the other constitutive promoters that were tested, so that its relative expression can be determined. This is useful when deciding which promoter to use for genetic engineering. Figure 3 shows the growth curves of the constitutive promoters.
Growth curves
The bacteria carrying the different mCherry expressing plasmids were expected to have similar growth curves. However, Figure 3 shows that presence of the PmxaF, PcoxB and Ptuf inhibited the maximum OD600nm the cultures reached, when compared to the control and the PfumC strain. For the measurements of fluorescence, generally the middle of the exponential phase of growth is taken [3]. In this experiment, the 24 hour sample was used for comparison in Figure 2.
Figure 3: Growth curves of the constitutive promoters over time of the mCherry expressing strains, sorted by promoter, showing the mean of the biological and technical duplicates, with the dotted lines representing the error bars. A sigmoidal 4PL curve (black) is fitted on each graph. The strains were grown over a timespan of 5 days in a plate reader. Similar growth rates are seen when comparing the no promoter strain to the PfumC strain. The other strains show a lower value for the stationary phase.
Fluorescence
Besides the absorbance, the fluorescence of mCherry was measured, shown in Figure 4. As expected, the strain with no promoter before the mCherry cassette shows almost no fluorescence. For the strains with the different promoters, the fluorescence reaches a maximum at 48 hours, which is to be expected, since the bacteria stop increasing in number at that time, as can be seen in Figure 3. The maximum fluorescence signal differs between the different promoters, indicative of a difference in promoter strength. Contrary to the expectation, one of the biological duplicates of PcoxB did not show fluorescence. This indicates an aberration suppressing mCherry expression in this specific biological duplicate, therefore the data from this duplicate was excluded from the analysis.
Figure 4: mCherry Fluorescence of the constitutive promoters over time of the mCherry expressing strains, sorted by promoter, showing the mean of the biological and technical duplicates, with the dotted lines representing the error bars. A sigmoidal 4PL curve (black) is fitted on each graph. The strains were grown over a timespan of 5 days in a plate reader. Similar fluorescence curves are seen between the constitutive promoter strains, however the values are different orders of magnitude, indicating different promoter strengths. The ‘no promoter’ strain hardly has a fluorescence signal, which is consistent with the expectations.
Fluorescence divided by OD
We plotted Fluorescence divided by OD600nm over time in Figure 5, thereby combining Figure 3 and 4. This step is taken to normalize the fluorescence signal by the amount of cells. This will compensate for the fact that a higher amount of cells will produce a higher fluorescence signal, thereby giving a clearer insight in promoter activity. This makes it possible to compare the different promoters and biological duplicates, taking the growth phase (see OD600nm figures) into account.
Figure 5: mCherry Fluorescence divided by OD600nm of the constitutive promoters over time of the mCherry expressing strains, sorted by promoter, showing the mean of the biological and technical duplicates, with the dotted lines representing the error bars. A sigmoidal 4PL curve (black) is fitted on each graph. The strains were grown over a timespan of 5 days in a plate reader. For the PfumC and PcoxB strains, the fluorescence/OD plateaus over time. The graph for the PmxaF keeps increasing, which can be attributed to a decrease in absorbance as seen in Figure 3. The ‘no promoter’ strain hardly has a fluorescence/OD signal, which is consistent with the expectations.
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