Part:BBa_K4721006

P_L/O4/A1 IPTG inducible system

This composite part functions as an IPTG inducible promoter. P_L/O4/A1 is a synthetic promoter designed by Carrillo et al. (2019)[1]. Figure 1 illustrates the part structure. In order to function as an inducible promoter, the lacI gene (BBa_K1222003)with its respective lacIq promoter (BBa_K4721005)is expressed, which expresses the lac repressor, LacI. This repressor binds the lacO sequence, present in the P_L/04/A1 in the interspacer region between the -10 and -33 sites (BBa_K4721004). When Isopropyl β-d-1-thiogalactopyranoside (IPTG) is present, this repressor is released, and the respective gene (here mCherry) is expressed.

Figure 1: Schematic overview of the mCherry reporter under the control of the composite P_L/O4/A1 promoter system

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 4
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 90
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 4
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 4
  
• 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
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)[2] 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 [3] 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.

Induction of P_L/O4/A1

From here on, we will discuss our results of P_L/O4/A1. The growth experiment was set up with 10 different IPTG concentrations ranging from 0 - 1,000µM. This concentration range was based on Carrillo et al. (2019)[1]

Growth curves

The bacteria carrying mCherry under the control P_L/O4/A1 were expected to have similar growth curves, unless the IPTG were toxic to the cells, or the metabolic burden of the plasmid would inhibit the growth. Figure 3 shows that indeed the growth curves are similar between the conditions. For the measurements of fluorescence, generally the middle of the exponential phase of growth is taken [3]. For this promoter, the 48 hour sample was used for comparison in Figure 2.

Figure 3: Growth curves of the different conditions for the strain expressing mCherry under the control of the P_L/O4/A1 promoter, and of the control without a promoter. IPTG concentration in µM is mentioned above each graph. The mean of the biological and technical duplicates is shown, 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 between the conditions.

Fluorescence

Besides the absorbance, the fluorescence of mCherry was measured, as shown in Figure 4. As expected, the strain with no promoter before the mCherry cassette shows almost no fluorescence. For the different conditions for the strain expressing mCherry under the control of the P_L/O4/A1 promoter, an increase in fluorescence with increasing concentration of IPTG can be seen, especially from 500µM IPTG onwards.

Figure 4: mCherry Fluorescence of the different conditions for the strain expressing mCherry under the control of the P_L/O4/A1 promoter, and of the control without a promoter. IPTG concentration in µM is mentioned above each graph. The mean of the biological and technical duplicates is shown, 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. The ‘no promoter’ strain hardly has a fluorescence signal, which is consistent with the expectations. Fluorescence increases with increasing IPTG concentrations.

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.

Figure 5: mCherry Fluorescence divided by OD_600nm of the different conditions for the strain expressing mCherry under the control of the P_L/O4/A1 promoter, and of the control without a promoter. IPTG concentration in µM is mentioned above each graph. The mean of the biological and technical duplicates is shown, 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. The ‘no promoter’ strain hardly has a fluorescence signal, which is consistent with the expectations. Fluorescence increases with increasing IPTG concentrations. Because of the normalization, this increase in signal can be attributed to accumulation of fluorescent protein in each cell, and not because of more cells.

Dose-response

Lastly, in Figure 6, the 48h sample of the fluorescence/OD is plotted against the IPTG concentrations in µM. This figure shows the dose-response curve of the separate biological duplicates. There is a base fluorescence signal with no IPTG added, this indicates expression in the uninduced condition. Nonetheless, increasing inducer concentrations resulted in higher expression when comparing the uninduced condition with the 1000µM IPTG condition (190% increase, Student’s T-test, **** p < 0.0001).

Figure 6: Normalized expression of mCherry as function of IPTG concentration under control of the P_L/O1/A1 promoter to different IPTG dosages, separated over the two biological duplicates, of the 48h sample. Symbols represent the mean, and the error bars the standard deviation. The curves are fitted as Dose-response - Stimulation; [agonist] vs. response (three variables) using GraphPad Prism. Fluorescence/OD is relatively high with no IPTG added, but with increasing concentrations the fluorescence/OD increases.