Notebook - September/October 2025

From 15/09/25 to 07/10/25 - Experiments conducted by Maëva Chopis and Elisa Afonso.

Objectives

Purify RPA1163 (A1), RPA1163 XR1 (O1, a RPA1163 mutant) and Lipase SpL (U1) enzymes (control is the WT MG1655 strain, referenced as WT).
Reproduce the results reported in the literature for the colorimetric test that monitors defluorination by pH.
Test this method on our enzyme and cyclic TFA.
Expose Lipase (U1) to TFA for further investigation.

Notes

All following cultures were performed in LB medium, as TB medium promotes vesicle formation, which was no longer desired.

A. Experiments

Experiment 1: OD Monitoring

15/09/25

Purpose:

Compare growth curves between the wild type strain MG1655, and MG1655 A1 in which the gene encoding RPA1163 WT (named A1) was inserted.

Methods and Procedures:

"Protocol: Bacterial culture - Optical density and protein quantity monitoring" (see experiment section) with WT and A1
"Protocol: SDS-Page." SDS-Page gel visualization with a sample loaded at an optical density unit (ODu) of 0.02 per well.

To compute the volume of cracking buffer necessary to reach 0.02 ODu, this computation was made :

ODu well = 0.02 ODu V well = 10 µL C well = 2 ODu/mL ODu pellet = measured V buffer = (ODu pellet / C well) \times 10 3 µL

Results:

Figure 1: OD monitoring of MG1655 WT and A1 cultures

	SP			A1
Time (hours)	ODu	Cracking buffer volume (mL)	ODu/mL	ODu	Cracking buffer volume (mL)	ODu/mL
0	0,1	0,05	2	0,11	0,055	2
1	0,19	0,095	2	0,16	0,08	2
1,5	0,36	0,18	2	0,24	0,12	2
2	0,66	0,33	2	0,41	0,205	2
2,5	0,91	0,455	2	0,73	0,365	2
3	1,27	0,635	2	1,08	0,54	2
3,75	1,32	0,66	2	1,58	0,79	2
4,25	1,32	0,66	2	1,52	0,76	2
5	1,66	0,83	2	1,82	0,91	2
5,5	1,88	0,94	2	2,56	1,28	2
6,75	2,52	1,26	2	3,16	1,58	2
7,25	3,84	1,92	2	4,44	2,22	2
9	3,92	1,96	2	5,28	2,64	2
Total volume	11,085			12,695

Table 1: Calculation of the cracking buffer volume to add so that each SDS-PAGE well contains the same amount of sample (normalized to OD units) and OD monitoring as a function of time (hours). The red values seemed aberrant.

Observations and Notes:

Contamination was suspected because the culture never reached the stationary phase.
Nothing was visible on the SDS-PAGE gels; one hypothesis is that 0.02 OD units per well was not sufficient.
Another possibility is that there was an issue with sample preparation or gel staining.
In the following gels, the APS solution and the Coomassie blue solution were freshly prepared, and the staining and destaining steps were carried out overnight instead of 30 minutes.

17/09/25

Purpose:

Verify previous growth results and compare growth curves between MG1655 WT, MG1655 A1 and MG1655 U1 in which the gene encoding Lipase (named U1) was inserted.

Methods and Procedures:

"Protocol : Bacterial culture - Optical density and protein quantity monitoring" (see experiment section) with MG 1655 WT, MG1655 A1 and MG1655 U1.
"Protocol : SDS-Page." SDS-PAGE gel was revealed using 0.4 OD units per well.

Results:

The initial OD values of the starter cultures were as follows:

WT (MG1655 without plasmid): OD = 3.12
A1: OD = 3.28
U1: OD = 2.08

To standardize the initial OD to approximately 0.03 in each final culture, the following volumes of starter culture were pipetted:

WT: 0.96 mL
A1: 0.91 mL
U1: 1.44 mL

Figure 2: OD monitoring of MG1655 WT, A1 and U1

Number	Time	A1			U1			WT
( )	(hours)	A1	Cracking buffer volume (µL)	ODu/mL	U1	Cracking buffer volume (µL)	ODu/mL	WT	Cracking buffer volume (µL)	ODu/mL
0 (9h)	0	0,02	0,5	40	0,02	0,5	40	0,02	0,5	40
1	1,17	0,1	2,5	40	0,1	2,5	40	0,09	2,25	40
2	1,75	0,16	4	40	0,27	6,75	40	0,21	5,25	40
3	2,25	0,25	6,25	40	0,45	11,25	40	0,5	12,5	40
4	2,75	0,42	10,5	40	0,48	12	40	0,52	13	40
4bis	3	0,42	10,5	40	0,48	12	40	0,52	13	40
5	3,25	0,58	14,5	40	0,66	16,5	40	0,72	18	40
6	4,75	0,89	4,45	40	0,91	4,55	40	1,04	5,2	40
7	5,25	1,42	35,5	40	1,38	34,5	40	1,44	36	40
8	6,5	2,04	51	40	1,92	48	40	2,12	50	40
9	7,25	1,68	42	40	1,68	42	40	2	50	40
10	8	2,08	52	40	1,92	48	40	2,08	52	40
12	22,5	2,6	13	40	3	15	40	2,7	13,5	40

Table 2: Calculation of the cracking buffer volume to add so that each SDS-PAGE well contains the same amount of sample (normalized to OD units) and OD monitoring as a function of time (hours)

Each culture were induced around 0.5, respectively at 0.58, 0.48 and 0.5 for A1, U1 and WT.

Each number on the SDS-Page gels corresponds to a time point (from 1 to 12). The correspondence can be found in the table above.

Figure 3.1: SDS-PAGE monitoring of our WT culture between 2:45 and 22:30 of growth. The sample numbers correspond to the numbers in the previous table.

Figure 3.2: SDS-PAGE monitoring of our A1 culture between 2:45 and 22:30 of growth. The sample numbers correspond to the numbers in the previous table.

Figure 3.3: SDS-PAGE monitoring of our U1 culture between 2:45 and 22:30 of growth. The sample numbers correspond to the numbers in the previous table.

Observations and Notes:

The growth curves are more consistent and show similar profiles. Thus, the production of our enzymes has no impact on the development of bacteria.
Two hypotheses arise from this curve. First, the production of A1 and U1 enzymes does not appear to be toxic to the bacteria, as their growth curves are similar to that of the wild type (WT). Second, due to the presence of the Vnp sequence, the enzymes may in fact be toxic, but this effect is not visible in the growth curve because the enzymes are not retained in the cytoplasm, they are instead exported in vesicles.
On the SDS-PAGE gel, enzyme overproduction is visible at 5:15 for all cultures, which aligns well with the induction time of approximately 4:45. While the enzyme quality remains undetermined, their quantity appears to reach a maximum from 7:15 onwards. No protein overproduction is observed for MG1655 WT, which is consistent with its role as the control. We therefore maintained our induction when the OD reached 0.5 and the culture overnight.

Experiment 2: Sonication Tests

19/09/2025

Purpose:

Determine the optimal sonication conditions to efficiently lyse bacteria without compromising enzyme integrity.

Methods and Procedures:

"Protocol: Bacterial culture - Enzyme overproduction culture" of A1 induced and non-induced from 15/09/25-18/09/25 (culture n°1)
"Protocol: Purification of the cell pellet" with option 2 (sonication) and until step 3.

Different sonication conditions were tested (the changing conditions are in bold), as follows:

50 mL pellet resuspended in 5 mL of PBS, 10 cycles of 10 pulses at power 8, duty cycle 30%
250 mL pellet resuspended in 25 mL of PBS, 10 cycles of 10 pulses at power 8, duty cycle 30%
50 mL pellet resuspended in 5 mL of PB, 10 cycles of 10 s continuously at power 10, duty cycle 30%
50 mL pellet resuspended in 5 mL of PB, 10 cycles of 10 pulses at power 4, duty cycle 30%
50 mL pellet resuspended in 5 mL of PBS, 10 cycles of 10 pulses at power 8, duty cycle 50%
100 mL pellet (07/09) resuspended in 10 mL of PBS, 10 cycles of 10 pulses at power 8, duty cycle 30%
100 mL pellet (07/09) resuspended in 5 mL of PBS, 10 cycles of 10 pulses at power 8, duty cycle 30%
100 mL pellet (07/09) resuspended in 10 mL of PBS, Without sonication
50 mL pellet resuspended in 5 mL of Cell lytic

There is a 30-second pause between each cycle.

"Protocol: SDS-Page"

Results:

A dilution gel was first prepared to determine the optimal sample dilution for the subsequent SDS-PAGE, which will be used to identify the best experimental condition.

Figure 4: SDS-Page of dilution tests for an SDS-PAGE of sonication tests. The conditions tested are as follows: 1. Condition 1 of the undiluted sonication test, 2. Condition 1 diluted 10 times, 3. Condition 1 diluted 100 times, 4. Condition 2 undiluted 5. Condition 2 diluted 10 times 6. Condition 2 diluted 100 times 7. Condition 7 diluted 10 times.

The wells with the most readable staining and the clearest results are wells 1 and 4, which correspond to the undiluted samples. We therefore decided to perform SDS-Page to compare sonication conditions without diluting the samples.

Figure 5: SDS-PAGE of sonication tests. The tested conditions are described in the Methods section above

Observations and Notes:

There is no visual difference between conditions 1, 2, 4, and 5 on our gel. We can therefore conclude qualitatively that increasing the sonication volume fivefold and reducing the power had no observable impact.
Condition 3 seems promising because the total protein amount decreases while the target enzyme is still produced, but overheating was observed during the process, which may have denatured our enzymes.
Between conditions 6 and 8, there is also no visual difference, even though they come from the same culture and condition 8 was not sonicated.
For the next experiments, we will reduce the number of cycles to 5 and centrifuge 250 mL of culture in the same Falcon tube to save time on sonication, since it is time-consuming and prevents us from handling other experiments in parallel.
The last well corresponds to CellLytic lysis and appears quite similar to wells 1, 2, 4, and 5, suggesting no major visual difference between lysis using CellLytic and lysis via sonication combined with lysozyme.

Experiment 3: A1 and U1 Purification - Cell Lysis Protocol Comparison

22/09/25 - 24/09/25

Purpose:

Compare two cell lysis protocol in purification enzyme production: sonication and cell lytic.

Methods and Procedures:

"Protocol: Purification of the cell pellet" with option 1 (cell lytic) or option 2 (sonication)
"Protocol: SDS-Page"

Thawing two 100 mL culture cell pellets of the A1 culture from the 24/08 week. Purification of the cell pellet is performed entirely on the two Falcon tubes, but in step 2, one is subjected to option 1 (cell lytic) and the other to option 2 (sonication). For SDS-Page samples, 15 µL are taken at each stage and 5 µL of cracking buffer are added before heating. The amount of sample loaded was not the same for each well, so the band intensities cannot be directly compared. For example, the last fraction was concentrated in 250 µL, whereas the first was diluted in 50 mL.

Results:

Figure 6.1: SDS page of purification steps performed with cell lytic. The names of the wells correspond to samples from the different stages of bacterial pellet purification. They are described at the end of the notebook page.

Figure 6.2: SDS page of purification steps performed with sonication. The names of the wells correspond to samples from the different stages of bacterial pellet purification. They are described at the end of the notebook page.

Observations and Notes:

By comparing both gels, we could assume that the sonication purification protocol yielded more protein.
However, the first SDS-PAGE gel conducted during this internship revealed challenges related to sample preparation. Several samples contained less than the intended 10 µL prior to loading, which complicated band comparison and limited the reliability of the conclusions.
Nevertheless, it is certain that enzymes were lost during steps 4 and 5.1 of our protocol, corresponding to the unbound flow-through and the first resin washing step. We still find our enzyme around 33.692 kDa and TEV protease around 27 kDa.

24/09/25-26/09/25

Purpose :

Compare protein presence between clear lysate and insoluble pellet of WT, A1, RPA1163 modified 1 (named O1) and U1 with different conditions (with sonication, cell lytic or neither (only lysosyme). With these differents conditions, compare these enzymes at the end of the purification protocol.

Methods and Procedures:

"Protocol : Bacterial culture - Enzyme overproduction culture" for WT, A1, O1 and U1, culture n°3 (22/09/2025 - 24/09/2025).
"Protocol: Purification of the cell pellet" with option 1 or 2 or none (only lysozyme).

Cell Lysis Conditions for each (WT, A1, O1 and U1):

3.5 pellets lysed by sonication in 25 mL buffer
3.5 pellets lysed by sonication in 12.5 mL buffer
1 pellet without sonication (positive control)
1 pellet treated with Cell Lytic reagent (positive control)

"Protocol: SDS-Page"

Figure 7.1: SDS page of pellet (p) and clear lysate (s) of different cultures obtained after using Cell lytic

Figure 7.2: SDS page of pellet (p) and clear lysate (s) of different cultures obtained after Sonication

Figure 7.3: SDS page of pellet (p) and clear lysate (s) of different cultures obtained without sonication

The purified enzyme samples collected at the final stage are deposited on gel and then compared.

Figure 8.1: SDS-Page of protein purified by sonication (Son), with cell lysis (CL) and without treatment (WS)

Figure 8.2: SDS-Page of protein purified by sonication (Son), with cell lysis (CL) and without treatment (WS) + Test for a future SDS to see if the proteins would appear properly and if dilution was necessary before doing them all

Observations and Notes:

Each of our enzymes appears to be insoluble in the pellet. This is not what has been observed previously; a handling error or poor sample storage could be the cause. However, a greater amount of protein is found in the clear lysate with sonication than without sonication for all enzymes.
We obtain a much higher amount of enzymes with sonication than without (with lysozyme in both cases). The fact that there are none at all with cell lytic seems strange but confirms our observation on 21/08, which showed that it was less effective than sonication.
We will therefore apply the purification protocol with sonication (250 mL pellet resuspended in 25 mL of PBS, 10 cycles of 10 pulses at power 4, duty cycle 30 %) for numerous batches in order to obtain the maximum amount of purified enzymes.

Experiment 4: Protein Purification with Sonication Protocol

29/09/2025 - 01/09/25

Purpose:

Purification of WT, A1 and U1 with sonication lysis.

Methods and Procedures:

“Protocol: Bacterial culture - Enzyme overproduction culture” of MG1655 WT, MG1655 A1 and MG1655 U1 (culture n°4 - 23/09/25-26/09/25).
“Protocol: Purification of the cell pellet” with Option 2 of WT, A1 and U1. Two Falcon tubes containing 225 mL of pellet material were prepared for each culture.
“Protocol: SDS-Page”.
“Protocol: Protein Quantification ThermoScientific”.

Results:

The names of the wells correspond to samples from the different stages of bacterial pellet purification. They are described at the end of the notebook page.

Figure 10.1: SDS page of purification steps of MG1655 WT.

Figure 10.2: SDS page of purification steps of MG1655 A1

Figure 10.3: SDS page of purification steps of MG1655 U1

The concentration of the previous samples, as well as other previously purified ones, was measured using a BSA assay.

Figure 11: BSA calibration curve measured by spectrophotometry at 560 nm.

Table 3: Protein Quantification by BSA Assay: Standards, Absorbance Values (560nm), and Calculated Sample Concentrations. The samples for the first table come from enzymes purified on 24/09. The next two tables use lysate from 26/09, and the last table uses enzymes purified on 01/10.

First, the trend curve shows that the OD values at low concentrations are not very accurate. The measurement was taken 45 minutes after adding the reagent diluent, whereas a 30-minute incubation time is recommended. This may explain why the values obtained are inconsistent and difficult to interpret. For the clarified lysate (S) after sonication and CellLytic treatment, the control (WT) displayed higher values than the other samples, which was unexpected, since no proteins were visible on the gels. This suggests that other molecules may be interfering. In addition, in the first dataset, no control was available to calculate the true protein concentration. However, for the purified enzymes, the results appear more consistent, with concentrations above 1100 µg/mL for A1 and U1 but this cannot be confirmed based on the results of the other samples.

Figure 12: Determination of concentration in our pure enzyme samples with a range of BSA. The numbers above the wells correspond to the BSA concentrations of the sample in µg/mL.

BSA gel analysis with imageJ : BSA curve

Figure 13: BSA standard range - Band intensity as a function of time (correspond to the Figure 12 gel).

Band intensity	Protein concentration (µg/mL)
302,021	0
1532,82	25
4683,589	125
7118,167	250
12395,108	500
27043,141	750
33790,777	1000
62423,023	1500
97849,281	2000

Table 4: BSA standard range obtained with imageJ - (correspond to the Figure 10 gel)

Observations and Notes:

We did not obtain any purified enzyme according to our SDS-Page gels. For enzyme A1, no overexpression is visible from the start. For U1, a large amount is present in the clear lysate but nothing remains after the resin,this observation appears unexpected, since samples B4 and B5.1 did not exhibit a comparable level of loss. This is confirmed by the SDS-Page gel, on which a band can be observed between wells A1 and WT, but which corresponds to a staining artifact. Moreover, the gel we used had wells that were too narrow, which made interpretation more difficult.
A culture will be redone to achieve net overproduction of our enzymes, as well as the purification protocol, for which the loss of so much enzyme has not been explained. The BSA test will be carried out more rigorously, following the 30-minute incubation time.

01/10/25-03/01/10

Purpose:

Purify a subsequent protein quantity with the sonication lysis protocol.

Methods and Procedures:

“Protocol: Bacterial culture - Enzyme overproduction culture” of MG1655 WT, MG1655 A1, MG1655 O1, MG1655 U1 (culture n°6 - 29/09/25-01/10/25).
“Protocol: Purification of the cell pellet with Option 2” of WT, A1, O1 and U1.
“Protocol: Protein Quantification ThermoScientific”.
“Protocol: SDS-Page”.

Thawing pellets from -80°C from cultures of 01/09 and 07/09 weeks (OD not known).

In total, there are:

O1:

(1) 01/10: 1 falcon with a 250 mL pellet
(2) 01/09: 2 falcons with a 200 mL pellet
(3) 07/09: 5 falcons with a 300 mL pellet

A1:

(3) 01/10: 1 falcon with a 250 mL pellet
(1) 03/07: 1 falcon with a 300 mL pellet
(2) 07/09: 3 falcons with a 300 mL pellet

U1:

(1) 01/10: 1 falcon with a 250 mL pellet
(2) 01/09: 1 falcon with a 200 mL pellet and 1 with a 150 mL pellet

SP: 01/10: 1 falcon with a 250 mL pellet

Results:

For the culture of 29/09/25-01/10/25:

Objective: Achieve a final concentration of 40 OD units/mL in one Falcon tube.

Sample Sp - OD: 3.76 ODu/mL

50 mL culture: 188 ODu → Resuspend in 4.7 mL Tris + 94 µL lysozyme
200 mL culture: 752 ODu → Resuspend in 18.8 mL Tris + 376 µL lysozyme

Sample A1 - OD: 4.56 ODu/mL

50 mL culture: 228 ODu → Resuspend in 5.7 mL Tris + 114 µL lysozyme
200 mL culture: 912 ODu → Resuspend in 22.8 mL Tris + 456 µL lysozyme

Sample U1 - OD: 3.68 ODu/mL

50 mL culture: 184 ODu → Resuspend in 4.6 mL K-phosphate + 92 µL lysozyme
200 mL culture: 736 ODu → Resuspend in 18.4 mL K-phosphate + 368 µL lysozyme

Sample O1 - OD: 3.2 ODu/mL

50 mL culture: 160 ODu → Resuspend in 4 mL Tris + 80 µL lysozyme
200 mL culture: 640 ODu → Resuspend in 16 mL Tris + 320 µL lysozyme

This normalization to optical density units allows for direct comparison between samples, including the control.

Figure 14.1: SDS-PAGE of bacterial pellets (PL) and culture supernatants (SP)

The names of the wells correspond to samples from the different stages of bacterial pellet purification. They are described at the end of the notebook page.

Figure 14.2: SDS page of purification steps of MG1655 A1.1

Figure 14.3: SDS page of purification steps of MG1655 A1.2

Figure 14.4: SDS page of purification steps of MG1655 A1.3

Figure 14.5: SDS page of purification steps of MG1655 O1.1

Figure 14.6: SDS page of purification steps of MG1655 O1.2

Figure 14.7: SDS page of purification steps of MG1655 O1.3

Figure 14.8: SDS page of purification steps of MG1655 WT

Figure 14.9: SDS page of purification steps of MG1655 U1.1

Figure 14.10: SDS page of purification steps of MG1655 U1.2

Figure 15: SDS page of purified enzymes (A11, A12, A13, O13 and U11). The numbers 1, 2, 3, 4, and 5 correspond to the following BSA concentrations in µg/mL, respectively: 250, 750, 1500, 2000

BSA gel analyses with imageJ :

Figure 16: BSA standard range - Band intensity as a function of BSA concentration (µg/mL) - corresponds to the analysis of gel on Figure 15.

Band intensity	Protein concentration (µg/mL)
1300,891 BSA 1	25
5337,075 BSA 2	250
12895,894 BSA 3	750
21496,108 BSA 4	1500
22984,3 BSA 5	2000
20650,924 A11	1574,79382
23765,723 A12	1840,48617
21108,44 A13	1617,95135
21156,874 U13	1613,92615
19399,187 U11	1468,02065

Table 5: Correspondance band intensity computed with imageJ and protein concentration (µg/mL) - corresponds to the analysis of gel on Figure 15.

Figure 17: ImageJ analysis of the gel shown in Figure 15. Rectangles were drawn to isolate only the protein of interest (and not the TEV protease for example).. Within each selected region, band intensity was quantified using the “Plot Lanes” function in ImageJ, focusing exclusively on the relevant bands.

Absorbance BSA analysis - Protein Quantification ThermoScientific:

With the BSA assay analysis, we obtain a concentration (µg/mL) of:

A1.1: 678
A1.2: 437
A1.3: 228
WT: 0
O1.1: 0
O1.2: 20
O1.3: 1242
U1.1: 1223
U1.2: 0

Figure 18: BSA calibration curve measured by spectrophotometry at 560 nm.

Table 6: Protein Quantification by BSA Assay: Standards, Absorbance Values, and Calculated Sample Concentrations.

Observations and Notes:

For the enzymes A1.1, A1.2, A1.3, U1.1 and O1.3: We obtained purified enzymes with a slight presence of smaller enzymes, as we can see on these gels.
In almost all gels, following TEV addition, the TEV protease is co-eluted with the target protein.
Furthermore, a significant amount of protein is lost in the supernatant after incubation with Ni-NTA resin and the first wash step, indicating that a substantial fraction of the protein did not bind to the resin. This suggests that a larger volume of resin should have been used.
For the enzymes U1.1, O1.1, and O1.2: We did not achieve overproduction from the start, so we obtained little or no enzyme at the end of the purification.
The results obtained with BSA assay (absorbance assay) appear to be fairly consistent with what is observed on the gels. Indeed, O1.1, O1.2, and U1.1 do not contain our enzymes, and their protein concentrations are similar to that of WT. However, the unusually high protein concentration in WT is unexpected, and we were unable to determine its origin. Therefore, we subtracted this value to all other protein concentrations. However, these results seem quite inconsistent with those calculated with imageJ which gave higher protein production. Even if the latter is less precise, such a large difference is not explained. And no protein is seen on the WT gel,so as an enzyme concentration reference for the following experiments, we will take the values obtained from BSA quantification on SDS-PAGE gels were used for calculating enzymatic activity, as they appear to be more accurate.

Experiment 5: Soluble Protein Extraction

07/10/25

Purpose:

Obtain a clear lysate containing soluble proteins without further purification.

Methods and Procedures:

“Protocol: Bacterial culture: Enzyme overproduction culture” of MG1655 WT, MG1655 A1, MG1655 O1, MG1655 U1 (culture n°7 05/10/25-07/10/25).
“Protocol: Purification of the cell pellet with Option 2” of WT, A1, O1 and U1 until step 3. Bacterial pellets were resuspended in the corresponding activation buffer.
“Protocol: SDS-Page”.

Results:

Figure 19: SDS page of clear lysates (A1, O1, U1 and WT). The numbers 1, 2, 3, 4, and 5 correspond to the following BSA concentrations in µg/mL, respectively:, 25, 250, 750, 1500, 2000.

BSA analysis with imageJ :

Band intensity	Protein concentration (µg/mL)	Identifier
1410,841	25	1
7306,024	250	2
18654,522	750	3
27942,007	1500	4
36312,149	2000	5
2503,841	130,450116	A1
9102,853	474,258614	U1
244,263	12,7261023	O1
208,435	10,8594635	WT

Table 7: Correspondance band intensity computed with imageJ and protein concentration (µg/mL) - corresponds to the analysis of gel on Figure X.

Figure 20 : BSA standard range - Band intensity as a function of BSA concentration (µg/mL) - corresponds to the analysis of gel on Figure 19.

Observations and Notes:

Overproduction was unsuccessful for all enzymes, possibly due to the use of aged bacterial cultures.With the imageJ analysis, we reach a maximum enzyme quantity for U1 with 474 µg/mL of enzyme in the sampl. These quantities are not sufficient to do any analysis.

Experiment 6: Inductor Petri Dish and Culture

05/10/25

Purpose:

To visually quantify the defluorination activity of the RPA1163 enzyme. Based on the protocol described in (Holloway et al., 1998). This experiment was carried out to gain an initial overview and to test it on the entire bacterial culture.

Methods and Procedures:

“Protocol: Bacterial culture: Inductor petri dish culture”
Conditions : For MG1655 WT, MG1655 A1, MG1655O1, 100µL of substrate of 0, 10 and 100 mM of fluoracetate was adding. Each experiment was done in triplicate.

Results:

figure 21.1: Experiments conducted under PSM for the inductor petri dish

Figure 21.2: Inductor petri dish after a night incubation.

Figure 21.3: Representative inductor petri dish after 10 min of all conditions.

Observations and Notes:

Since fluoroacetic acid lowers the pH of the medium, a yellow halo appeared upon the addition of 100 µL of 100 mM fluoroacetic acid.
However, yellow is the color associated with low pH for both bromothymol blue and phenol red. As acidification of the medium is expected during the SN2 reaction, this observation alone does not allow any conclusion regarding enzymatic activity.
At a concentration of 10 mM, no yellow halo was observed, but the color remained unchanged compared to the control, indicating no detectable pH shift.
After overnight incubation, all plates had turned completely purple, indicating full alkalinization of the medium due to the presence of our bacteria—an effect we had not anticipated.
Based on the results obtained from this experiment, we considered repeating the experiment on plates, but with fluoroacetate directly incorporated into the solid medium and adjusting the color to be at the onset of the color change, followed by monitoring the plates every 10 minutes after inoculation.
We also planned to perform this experiment in liquid culture, including fluoroacetate directly in the medium and adjusting with NaOH so that samples with and without the acid would have the same color at time zero. Monitoring every 10 minutes was also intended.
While we were unable to carry out the first protocol as initially planned, the second protocol was successfully performed.

Experiment 7: Solubility Test

05/10/25

Purpose:

To assess the solubility of selected chemicals in water.

Methods and Procedures:

Chemicals tested:

Fluoroacetic acid (high polarity)
N-[(4-chlorophenyl)methyl]-2,2,2-trifluoroacetamide (cyclic TFA)

Each compound was initially dissolved in 100% water. If the solution appeared turbid, methanol was added incrementally until complete solubilization was achieved.

Results:

Solubility tests with methanol/water mixtures at 100 mM TFA:

10% methanol / 90% water → not soluble (heavy turbidity)
50% methanol / 50% water → not soluble (moderate turbidity)
62% methanol / 38% water at 90 mM → soluble

Prepared Solutions:

100 mM solution of fluoroacetic acid
90 mM solution of N-[(4-chlorophenyl)methyl]-2,2,2-trifluoroacetamide

Observations and Notes:

Solubility Assessment:

Fluoroacetic acid, as expected due to its high polarity, was fully soluble in water.
Cyclic TFA, at 90mM, was not soluble in pure water.

Experiment 8: Colorimetric Monitoring - Liquid Culture

06/10/25

Purpose:

Visually observe defluorination by colorimetry. A color change to yellow is expected because the defluorination of fluoroacetate by nucleophilic substitution by the enzyme releases H+ ions. This experiment was carried out to gain an initial overview and to test it on the entire culture.

Methods and Procedures:

“Protocol: Colorimetric monitoring - Liquid culture”

The conditions tested were as follows:

MG1655 WT, FA 0 mM
MG1655 WT, FA 2 mM
MG1655 WT, FA 4 mM
MG1655 A1, FA 0 mM
MG1655 A1, FA 2 mM
MG1655 A1, FA 4 mM
MG1655 O1, FA 0 mM
MG1655 O1, FA 2 mM
MG1655 O1, FA 4 mM

Each Erlenmeyer flask was induced with between 4 and 5 hours of culture.

Results:

Figure 22.1: Erlenmeyer flasks after adding the dye, FA solution, and adjusting the color (O1 0mM, O1 2mM, O1 4mM, WT 0mM, WT 2mM, WT 4mM, A1 0mM, A1 2mM, A1 4mM).

Figure 22.2: Erlenmeyer flasks after 2 hours of incubation (O1 0mM, O1 2mM, O1 4mM, WT 0mM, WT 2mM, WT 4mM, A1 0mM, A1 2mM, A1 4mM).

Observations and Notes:

No differences were observed between the flasks after 10 and 30 minutes of culture. After overnight incubation, a basic color was again observed in the medium due to the presence of the bacteria.

Experiment 9: Colorimetric Monitoring - Plate

The following protocols are inspired from (Holloway et al., 1998), (Khusnutdinova et al, 2023) and (Chan et al., 2011).

03/10/25

Purpose:

Quantify dehalogenases defluoration for different substrates concentration of fluroacetate using clear lysate.

Methods and Procedures:

“Protocol: Colorimetric monitoring - Plate with phenol red protocol using a TECAN. Clear lysates come from the soluble protein extraction of the 01/10/25-03/01/10 during purification protocol (from culture n°6).
In each well, 195µl de tampon/subtrat (185µl buffer/substrat + 10µl de rouge de phénol) and 5µl de lysat clair were added. Buffer : 1mM HEPES.

Conditions:

Three substrates concentrations : 0, 5 and 10mM of fluoroacetate and cycled TFA.

Results:

The experiment with cyclic TFA was abandoned due to its lack of solubility in water. When a solution containing methanol, water, and cyclic TFA was added, it became opaque, preventing further analysis.
For fluoroacetate, absorbance monitoring was performed; however, no interpretable results were obtained. The standard HCl range showed identical absorbance values regardless of concentration. This was because the solution in each well appeared yellow from the start of the experiment, regardless of the initial HCl concentration.

Observations and Notes:

Phenol red changes color with pH, transitioning from red to yellow. Since all wells were already yellow at the beginning of the experiment, any variation in HCl concentration or potential defluorination by enzymes could not be visually detected. This also indicates that the initial pH of the solution was too low.
Future experiments will be repeated using phenol red pre-diluted directly into the buffer, with pH adjusted beforehand to ensure that the standard curve produces a visible color change (540nm is in the visible range).

07/10/25

Purpose:

Quantify dehalogenases defluoration for different substrates concentration of fluroacetate.

Methods and Procedures:

“Protocol: Colorimetric monitoring - Plate” with phenol red protocol using a TECAN. A1.3 and O1.3 enzymes were used from the purification of the 29/09-01/09/25. Clear lysates come from the soluble protein extraction of the 07/10.

Experimental design

WT 0 mM	WT 0 mM	WT 0 mM	A1 0 mM	A1 0 mM	A1 0 mM	O1 0 mM	O1 0 mM	O1 0 mM	Standard 0	Standard 0	Standard 0
WT 5 mM	WT 5 mM	WT 5 mM	A1 5 mM	A1 5 mM	A1 5 mM	O1 5 mM	O1 5 mM	O1 5 mM	Standard 1	Standard 1	Standard 1
WT 10 mM	WT 10 mM	WT 10 mM	A1 10 mM	A1 10 mM	A1 10 mM	O1 10 mM	O1 10 mM	O1 10 mM	Standard 2	Standard 2	Standard 2
WT 0 mM	WT 0 mM	WT 0 mM	A1 0 mM	A1 0 mM	A1 0 mM	O1 0 mM	O1 0 mM	O1 0 mM	Standard 3	Standard 3	Standard 3
WT 5 mM	WT 5 mM	WT 5 mM	A1 5 mM	A1 5 mM	A1 5 mM	O1 5 mM	O1 5 mM	O1 5 mM	Standard 4	Standard 4	Standard 4
WT 10 mM	WT 10 mM	WT 10 mM	A1 10 mM	A1 10 mM	A1 10 mM	O1 10 mM	O1 10 mM	O1 10 mM	Standard 5	Standard 5	Standard 5
Standard 8	Standard 8	Standard 8	0 mM	0 mM	0 mM				Standard 6	Standard 6	Standard 6
5 mM	5 mM	5 mM	10 mM	10 mM	10 mM				Standard 7	Standard 7	Standard 7

Clear lysate - Fluoroacetate (FA)

Standard range

Purified enzyme - FA

Substrate (FA) without enzymes

Table 8: Experimental design

Fluoroacetate was used as the substrate at concentrations of 0, 5, and 10 mM for two enzymes, A1 and O1, with the wild-type (WT) bacterial sample serving as a control.

After computing the absorbance mean between each triplicate, to compute normalized absorbance for all enzymes, ensuring that A = 0 at t = 0 for all samples (including the standard curve), the following calculation was applied:

A_norm = A_sample − A_sample(t₀)

With this normalized absorbance, A_norm is expected to be 0 at [FA] = 0 mM, resulting in a y-intercept at (0, 0). The linear regression is therefore constrained to pass through the origin. The standard deviation on A_norm was computed as follows:

Std(A_norm) = √( std(A_sample)²⁄3 + std(A_blank)²⁄3 )

3 is the number of replicates.

Thanks to the HCl standard range, the link between the concentration of H+ in the medium and A_norm can be made, and is a linear curve passing trough (0,0) (e.g., A_norm = a*[H+]. The curve of the concentration of H+ freed in the medium as a function of time can be plotted. The standard deviation of the concentration can be computed as follows:

Std(concentration H⁺) = |a| × Std(A_norm)

As the relationship between both is linear.

Finally, the enzymatic activity can be computed as follows:

n(H⁺) ⁄ (t × m(E)) = V₀ × V_well ⁄ m(E), (µmol/min/mg).

Results:

Figure 23: Weakly buffered solution with 1mM HEPES and phenol red

Figure 24: HCl standard range from 1mM Hcl (yellow) to 0mM HCl (pink)

Figure 25: Normalized absorbance variation over time for different concentrations of fluoroacetate (0, 5 and 10mM) in clear lysate corresponding A1, O1, and WT MG1655. Curves obtained after a colorimetric assay.

Absorbance monitoring - Purified enzymes

Figure 26: Normalized absorbance variation over time for different concentrations of fluoroacetate (0, 5 and 10mM) in purified enzymes corresponding A1.3, O1.3, and WT MG1655. Curves obtained after a colorimetric assay with absorbance taken at 540nm.

Absorbance monitoring - A1 purified enzyme

Figure 27: Normalized absorbance variation over time for different concentrations of fluoroacetate (0, 5 and 10mM) in purified enzymes corresponding A1.3 (RPA1163). Curves obtained after a colorimetric assay.

Figure 28: HCl standard range - Normalized absorbance at 540 nm as a function of HCl concentration (mM).

H+ concentration over time for purified A1

Figure 29: H+ concentration as a function of time for purified A1 (RPA1163) with three fluoroacetate concentrations : 0, 5 and 10mM.

H+ concentration over time for clear lysate A1

Table 9 : Computation over time of the concentration of H+ in the wells thanks to the relation : [H+]=-6.5936*A_norm.

In each 5µL of each enzyme sample was added, as the enzyme concentration is of 1574,79382 µg/mL according to the BSA gel analysis with imageJ. So, 7.5µg of enzyme were added in each well.

At 5mM enzymatic activity (µmol/min/mg) : 0.055 µmol/min/mg
At 10mM enzymatic activity (µmol/min/mg) : 0.045 µmol/min/mg

With V0 speed respectively at :

V0 = 2.23 µM/min à 5mM
V0 = 1.81 µM/min à 10mM

Enzyme efficiency is maximal when time is proportional to product concentration, i.e., when the initial rate (V₀) remains constant. For the 10 mM substrate concentration, the reaction curve remains linear up to t = 1800 s, and for 5 mM, up to t = 3000 s. Accordingly, V₀ was calculated between t = 600 s and 1800 s for 10 mM, and between t = 600 s and 3000 s for 5 mM.

Observations and Notes:

The standard HCl calibration curve is quite linear (R² = 0.9898), confirming the expected relationship between concentration and absorbance at 540 nm.
In the soluble protein extraction performed on 07/10/25 (e.g., clear lysate), no overproduction was observed, and consequently, no enzymes were present in the medium. As anticipated, the results were not interpretable, and no defluorination was detected.
Regarding the O1 purified enzyme, tested at 0, 5, and 10 mM fluoroacetate, behaved similarly to the control. This was expected, as the enzyme was engineered to have an affinity for cycled TFA, which reduces its affinity for fluoroacetate (FA).
In contrast, acidification was observed with enzyme A1 at both 5 and 10 mM fluoroacetate. This result suggests that A1 is indeed active and capable of catalyzing the defluorination of fluoroacetate, consistent with findings reported in the literature. Khusnutdinova et al (2023) measured over 5.5 µmol/mg of enzymatic activity for RPA1163 (A1) at 5 mM fluoroacetate during 2 hours, so between 0.045 and 0.05 µmol/min/mg. The results we found are then consistent with the literature.
The only difference is that we used 20 µg of enzyme per well. In our experiment, as the enzyme quantity was unknown at the experiment time, we used 5 µL of enzyme solution, the same volume used for the clear lysate, which is not optimal. Although enzymatic activity does not necessarily scale with enzyme concentration, a follow-up experiment using a higher enzyme amount could be performed to recalculate the activity more accurately.
Finally, the purification protocol using sonication lysis does not appear to impair enzyme function, as the enzymes retained their ability to degrade their natural substrate as the same enzymatic activity as the literature.

Conclusion:

Over the course of this month of laboratory work, we successfully achieved several key objectives. First, we monitored bacterial cultures to assess whether our enzyme exhibited toxicity toward the host strain and to determine the point of maximal expression. We then purified our enzymes in large quantities by establishing and optimizing a purification protocol involving sonication. In addition, we tested different colorimetric assays to monitor the degradation of fluoroacetate, including one for which our results were consistent with previously published data.

The next steps would involve confirming the reproducibility of this experiment and extending the assays to enzymes specifically designed for other PFAS, notably cyclic TFA. We also possess a fluorogenic probe that we aim to test in clarified lysates, where the enzyme concentration seemed important when overproduction was present.

B. Cultures

Culture 1: A1 MG1655 culture (induced and non-induced)

15/09/25-18/09/25

Purpose:

To overproduce protein A1. A non-induced culture was used as a control.

Methods and Procedures:

Protocol 1: Enzyme overproduction culture of A1, induced and non-induced with aTc at the final concentration of 200ng/mL, for a 500 mL culture volume.
Each 50 mL culture pellet was collected and stored in a 50 mL Falcon tube.

Results:

Induction of the A1 culture was performed at an OD of 0.56.
Final OD of the induced culture: 2.00.
Final OD of the non-induced culture: 1.91.

Culture 2: MG1655 culture without plasmid (control)

16/09/25 - 18/09/25

Purpose:

To have a negative control (WT strain) for the following manipulations.

Methods and Procedures:

Protocol 1: Enzyme overproduction culture of WT MG1655 for two 500mL culture volumes.

Culture 3: A1, U1, O1 and WT MG1655 cultures

22/09/2025 - 24/09/2025

Purpose:

To overproduce protein A1, U1 and O1.

Methods and Procedures:

Protocol 1: Enzyme overproduction culture of A1, U1, O1 and WT MG1655 for a 500mL culture volume each.

Results:

For final cultures (in 5L flask), 4 mL of each starter culture was added. The measured optical densities (OD) of the starter cultures were as follows:

U1: 1.68
WT: 2.48
O1: 1.12
A1: 2.72

Cultures were induced with aTc at the final concentration of 200ng/mL at the following OD values:

WT: 0.47
A1: 0.75
U1: 0.55
O1: 0.65

Final ODs:

WT: 3.56
A1: 3.60
U1: 2.60
O1: 3.48

Culture 4: A1, U1 and WT MG1655 cultures

23/09/25-26/09/25

Purpose:

To overproduce protein A1 and U1.

Methods and Procedures:

Protocol 1: Enzyme overproduction culture of A1, U1 and WT MG1655 for a 500mL culture volume each.

Results:

The optical densities (OD) of the starter cultures were as follows:

WT: 1.28
A1: 1.20
U1: 1.04

To achieve a starting OD of 0.01 in each final culture (500 mL in 5L flask), the following volumes of starter culture were calculated:

WT: 3.90 mL
A1: 4.20 mL
U1: 4.80 mL

Since the calculated volumes were very similar, 4 mL of each starter culture was ultimately added. Since it was too late to induce the cultures in the evening, they were incubated overnight at 18°C. The following morning, the optical density (OD) reached 0.35 for all cultures, and all three final cultures were induced at this OD.

Culture stopped when each culture reached respectively an OD of:

WT: 2.16
A1: 2.00
U1: 1.92

Culture 5: A1, U1, O1 and WT MG1655 cultures

24/09/25-26/09/25

Purpose:

To overproduce protein A1, U1 and O1.

Methods and Procedures:

Protocol 1: Enzyme overproduction culture of A1, U1, O1 and WT MG1655 for a 500mL culture volume each.

Results:

Induction was performed late, at the following optical densities (OD):

A1: 1.20
U1: 1.20
WT: 1.12
O1: 0.96

Final OD Measurements:

WT: 3.76
A1: 4.56
U1: 3.68
O1: 3.20

Culture 6: A1, U1, O1 and WT MG1655 cultures

29/09/25-01/10/25

Purpose:

To overproduce protein A1, U1 and O1.

Methods and Procedures:

Protocol 1: Enzyme overproduction culture of A1, U1, O1 and WT MG1655 for a 250 mL culture volume each.

Results:

After putting 5mL of each starter culture in each 250mL culture, the ODs were at:

U1: 0.04
O1: 0.02
WT: 0.03
A1: 0.02

ODs at induction:

A1: 0.51
WT: 0.56
U1: 0.43
O1: 0.53

Final ODs:

A1: 4.56
U1: 3.52
O1: 3.36
WT: 3.44

Culture 7: A1, U1, O1 and WT MG1655 cultures

05/10/25-07/10/25

Purpose:

To overproduce protein A1, U1 and O1.

Methods and Procedures:

Protocol 1: Enzyme overproduction culture of A1, U1, O1 and WT MG1655 for a 500mL culture volume each.

Results:

ODs at induction:

A1 OD: 0.49
O1 OD: 0.52
U1 OD: 0.54
WT OD: 0.48

Final ODs:

A1 OD: 4.16
O1 OD: 3.2
U1 OD: 3.36
WT OD: 3.44

C. Sample Names SDS-Page

B1.1 - Total culture
B1.2 - Resuspended pellet
B2 - Total lysate
B3.1 - Insoluble pellet
B3.2 - Soluble supernatant
B4 - Flow-through
B5.1 - Washing
B5.2 - Elution
B6 - After dialysis, purified enzymes

References

Chan, P. W. Y., Yakunin, A. F., Edwards, E. A., & Pai, E. F. (2011). Mapping the Reaction Coordinates of Enzymatic Defluorination. Journal Of The American Chemical Society, 133(19), 7461‑7468. https://doi.org/10.1021/ja200277d

Khusnutdinova, A. N., Batyrova, K. A., Brown, G., Fedorchuk, T., Chai, Y. S., Skarina, T., Flick, R., Petit, A., Savchenko, A., Stogios, P., & Yakunin, A. F. (2023). Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases. FEBS Journal, 290(20), 4966‑4983. https://doi.org/10.1111/febs.16903

Holloway, P., Trevors, J. T., & Lee, H. (1998). A colorimetric assay for detecting haloalkane dehalogenase activity. Journal Of Microbiological Methods, 32(1), 31‑36. https://doi.org/10.1016/s0167-7012(98)00008-6