PcoxB

This part has been described by Schada Von Borzyskowski et al. (2015)[1]. The source of this part is the putative promoter region of the coxB gene, coding for the cytochrome c oxidase subunit II (MexAM1_META1p3474) in M. extorquens AM1.

Figure 1: Schematic overview of the mCherry reporter under the control of the PcoxB promoter

Part Info

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 P_L/O4/A1 and vanillate inducible promoter P_V10. 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
P_L/O4/A1 IPTG inducible system	BBa_K4721006	P_L/O4/A1	BBa_K4721004	Promoter, Operator
		lacIq promoter	BBa_K4721005	Promoter
		lacIq gene	BBa_K1222003	Coding
		lacIq terminator	BBa_K4721007	Terminator
P_V10 vanillate inducible system	BBa_K4721008	VanR repressor gene	BBa_K4721009	Coding
		RBS to combine with P_bla-mut1T promoter	BBa_K4721013	RBS
		P_bla-mut1T	BBa_K4721012	Promoter
		P_V10	BBa_K4721011	RBS, Operator
		RBS to combine with P_V10	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 Tc^R. 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 P_L/O4/A1 and the vanillate inducible promoter P_V10 show significantly increased fluorescence when induced, where the induction of the IPTG inducible promoter P_L/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 P_L/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 P_V10 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 OD_600nm 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 OD_600nm was calculated using the M. extorquens specific formula: OD_600nm = (absorption in well - 0.0755)/0.2344. The fluorescence, OD_600nm and fluorescence/OD_600nm 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 PcoxB 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 OD_600nm 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 OD_600nm 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 OD_600nm figures) into account.

Figure 5: mCherry Fluorescence divided by OD_600nm 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.

Part:BBa_K4721001