Team:Thessaly/Contribution

Characterization

Overview

The Monitoring System of Amalthea comprises three separate modules. We chose to extensively characterize the Prom Module before working with it for a proof of concept experiment. Briefly, the Prom Module is a NOT-GATE device that is activated in the absence of Short-Chain Fatty Acids (SCFAs). Its sensing element is a SCFA-inducible promoter, pFliC, which is mainly activated by butyrate. Since there is structural similarity to SCFAs butyrate, acetate and propionate, we sought to characterize the function of pFliC using all three and assess the promoter’s suitability in our project.

Aim

To add documentation data on promoter FliC (BBa_K2924016) and use that information for the development of our proof of concept, we run a Plate Reader Assay with different conditions. We used E. coli strain MC1061, as it is the workhorse chassis for our system. Since pFliC characterization using butyrate was done by Team Duesseldorf 2019, we tested the function of pFliC using acetate and propionate, which we are also interested in for our project.

Constructs’ Engineering

We evaluated pFliC’s function using three reporter genes, eCFP, eGFP, and sfGFP to provide a more comprehensive characterization. For this purpose, we designed three different constructs, as indicated in Table 1. Moreover, we tested pFliC using a range of concentrations spanning five orders of magnitude.

Table 1. The Registry Parts used for the generation of our designed Biobricks for our characterization experiments.

Registry Parts used	Biobrick Name	Links to the Registry Page
BBa_K2924016	Promoter FliC	http://parts.igem.org/Part:BBa_K2924016
BBa_K3505017	Double terminator	http://parts.igem.org/Part:BBa_K3505017
BBa_K3505019	eGFP	http://parts.igem.org/Part:BBa_K3505019
BBa_K3505018	sfGFP	http://parts.igem.org/Part:BBa_K3505018
BBa_K3505020	eCFP	http://parts.igem.org/Part:BBa_K3505020
BBa_K3505028	pFlic:RBS:eCFP:terminator	http://parts.igem.org/Part:BBa_K3505028
BBa_K3505029	pFlic:RBS:eGFP:terminator	http://parts.igem.org/Part:BBa_K3505029
BBa_K3505030	pFlic:RBS:sfGFP:terminator	http://parts.igem.org/Part:BBa_K3505030

Our three constructs contain parts that were ordered from IDT or were incorporated by PCR as overhanging sequences to primers annealing to the ordered part. We ordered the terminator we used, the constructs pFlic:RBS as one and we used PCR reaction for our reporter genes. After the successful PCR reaction, we used our pSEVA23 vector and we transformed it into E. coli DH5a cells (Figure 1). After verifying that we had our constructs in order, we transformed them into MC1061 for protein expression.

Figure 1. The results obtained after the PCR reaction for our three different devices. We expected bands in bp 3016 and 816 and the ladder that we used was the 100bp DNA ladder by NEB, in order to verify that the ligation was successful. It is demonstrated in Figures 1a, 1b, and 1c that we have accomplished the assembly of the three constructs.

Method

To produce reliable data we included 3 controls in our experiments.

Control 1: Untransformed cells

This gives us the background fluorescence output which is calculated by growing cells without eCFP, eGFP, or sfGFP (eCFP, eGFP, or sfGFP -less) in the same medium and microplate. The measured eCFP, eGFP or sfGFP -less fluorescence is subtracted from the final fluorescence output of transformed cells (with eCFP, eGFP or sfGFP ). 

Control 2: Empty wells

This gives the absorbance of the plate itself. This measurement is going to be subtracted from the final measurement of our construct.

Control 3: Wells with medium and antibiotic

M9 medium with antibiotic will give us the background fluorescence output. M9 medium does not emit fluorescence and is transparent.

The experimental workflow was as follows:

Plate Reader Assay 

1. Preparation of media required.

2. Complete the transformation with pFliC-eCFP, pFlic-eGFP, pFlic-sfGFP to MC1061.

3. Pick 3 colonies from each of these 3 plates. 

4. Put each colony into 5mL of LB pre-cultures and incubate O/N, 37oC, and 210 rpm.

5. Next morning, measure the OD600 of the overnight cultures.

6. For each one of the colonies: Dilute the culture to target OD600=0,1  in 5mL of desired medium and antibiotic. The number of M9 cultures depends on the time points you are going to choose.

7. Measure the OD600 and F1: 0h, 4h,8h,20h.
 
8. Add proper antibiotic (in this case, Kanamycin).

9. Incubate each culture: 37oC and 210 rpm. The duration of incubation varies for each liquid culture.

10. In due time: take 600 μl of each liquid culture and split into 3 walls ( 200μl per walls, technical replicates ).

11. Set the optimized parameters and measure.

Preparation of the 96-well plate with all additives

● Beginning of the plate-reader assay

● Timepoints: 0, 4h, 8h, 20h

● During these time points, the samples are in a shaker incubator.

● eCFP

- OD600

- Excitation at 434

- Emission at 477

● eGFP

- OD600

- Excitation at 488

- Emission at 515

● sfGFP

- OD600

- Excitation at 485

- Emission at 510

Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates.
The results of the assay can be seen in the figures below:

Figure 2. Adding acetate enabled the expression of eCFP, as pFliC is a SCFA-inducible promoter. We measure emission at 475nm, adding different concentrations of acetate. Our time-points were 0, 4, 8, and 20 hours, while we incubated each culture at 37oC and 210 rpm.

Figure 3. Different concentrations and time-points (0, 4, 8 and 20 hours) of acetate affect cell growth. As time passes there is an increase in the population of bacteria, but as there is an increase of the concentration of the acid, adding 20mM and 200mM acetate seems to prevent cell growth.

Figure 4. Adding propionate enabled the expression of eCFP, as pFliC is a SCFA-inducible promoter. We measure emission at 475nm, adding increasing concentrations of propionate.. Our time-points were 0, 4, 8 and 20 hours, while we incubated each culture at 37oC and 210 rpm.

Figure 5. Different concentrations and time-points (0, 4, 8 , 20 hours) of acetate affect cell growth. As time passes there is an increase in the population of bacteria, but as there is an increase of the concentration of the acid, adding propionate seems to affect cell growth.

Figure 6. Adding acetate enabled the expression of eGFP, as pFliC is a SCFA-inducible promoter. We measure emission at 488nm, adding increasing concentrations of acetate. Our time-points were 0, 4, 8 and 20 hours, while we incubated each culture at 37oC and 210 rpm.

Figure 7. Different concentrations and time-points (0, 4, 8 and 20 hours) of acetate affect cell growth. As time passes there is an increase in the population of bacteria, but as there is an increase of the concentration of the acid, adding acetate seems to prevent cell growth.

Figure 8. Adding propionate enabled the expression of eGFP, as pFliC is a SCFA-inducible promoter. We measure emission at 488nm, adding increasing concentrations of propionate. Our time-points were 0, 4, 8 and 20 hours, while we incubated each culture at 37oC and 210 rpm.

Figure 9. Different concentrations and time-points of acetate affect cell growth. As time passes (0, 4, 8 and 20 hours), there is an increase in the population of bacteria, but as there is an increase of the concentration of the acid, adding acetate prevents bacteria to grow.

Figure 10. Adding acetate enabled the expression of sfGFP, as pFliC is a SCFA-inducible promoter. We measure emission at 485nm, adding increasing concentrations of acetate. Our time-points were 0, 4, 8 and 20 hours, while we incubated each culture at 37oC and 210 rpm.

Figure 11. Different concentrations and time-points (0, 4, 8 and 20 hours) of acetate affect cell growth. As time passes there is an increase in the population of bacteria, but as there is an increase of the concentration of the acid, adding 20mM and 200mM acetate seems to prevent cell growth.

Figure 12. Adding propionate enabled the expression of sfGFP, as pFliC is a SCFA-inducible promoter. We measure emission at 485nm, adding increasing concentrations of propionate. Our time-points were 0, 4, 8 and 20 hours, while we incubated each culture at 37oC and 210 rpm.

Figure13. Different concentrations and time-points of acetate affect cell growth. As time passes (0, 4, 8, and 20 hours) there is an increase in the population of bacteria, but as there is an increase of the concentration of the acid, adding 200mM propionate seems to decrease cell growth.

The graphs above indicate that adding acetate and propionate can provoke the expression of the reporter genes. However, based on the concentration and the time-point that the measurement is taken, the results change. As time passes, the expression of each fluorescent protein is higher. Furthermore, there is the maximum expression of these three reporter genes, when we add 2mM acetate or propionate accordingly,  while it is indicated that adding increasing (20mM, 200mM) concentration of acids and, especially, acetate prevents cell growth. That may occur, due to their toxicity to the cells which may lead to unsettling results.