Team:FCB-UANL/Engineering

FCB:UANL Synbiofoam

 



FIRST CYCLE
SECOND CYCLE
THIRD CYCLE
FOURTH CYCLE
WESTERN BLOTS
PURIFICATIONS
FOAM TESTS
MODELS
FUTURE
REFERENCES
This year, we divided our wet lab efforts into two important stages. First, we developed the molecular protocols needed for transforming bacteria with our synthetic DNA (for further explanation go to our results section ). Then, we engaged into a cycle of continuous Learning and improvement of the expression of our proteins in our transformed bacteria. The complete laboratory protocols used this year can be found in the next file.

After we successfully transformed three E. coli Top 10 strains with the vector pSB1C3 containing Rsn-2 (BBa_K3498009 ) and Rsn-3-5 (BBa_K3498010 , BBa_K3498011 , BBa_K3498012 ) DNA respectively (for further information of the parts visit our project description section , and for the mentioned process visit our results section ), the second part of our biofoam creation was the Ranaspumins production and characterization. This step was a complete new experience for us, where every experiment and its analysis represented a new knowledge. According to the results of the experiments and our models' predictions, we adjusted the experiments and started over several times, fulfilling the engineering cycle.

Our main objective was to produce the core components of firefighting biofoam by successfully transforming E. coli Top 10 with the vectors containing our Ranaspumin DNA genes Rsn-2 and Rsn-3-5, and achieving their production. However, we also aimed to determine the best conditions for protein expression, using both models and experimental results (more details are available in our model section ) and test out the effectiveness of our firefighting biofoam.

Here we report, step by step, the whole process for constructing our foam: from the clonation and molecular techniques, going through the characterization of the Ranaspumins induction, and finally testing out the foam properties for its proposed implementation section .

FIRST CYCLE (Rsn-2 pSB1C3)

FIRST ITINERATION: DESIGN

Last year, the expression model of the FCB-UANL team 2020 (1) had already predicted that to have an optimal expression of Ranaspumins, a plasmid of low number of copies was necessary. In order to validate these results, we Designed the first cycle of experiments.

Aiming to express Rsn-2, we inserted the gene in our vector of high number of copies pSB1C3. According to the protocol described by Meyer and his collaborators (2), we inoculated with 1 mL of an overnight E. coli Top 10 culture in 100 mL of LB medium, adding a final concentration of vanillic acid of 100 mM. This culture was grown for 2 hours at 200 rpm and 37°C.

BUILD

The induced culture was incubated at 37°C with constant agitation at 200 rpm and 37°C for 12 hours.

TEST

We collected a 1mL sample every two hours for analysis in SDS-PAGE gel, which is shown in the next image.


LEARN

We observed there was no Rsn-2 expression, which validated the predictive model mentioned above. Thus, we needed to repeat the experiment using our vector of low number of copies: pSB3K3.

SECOND CYCLE (Rsn-2 pSB3K3)

FIRST ITINERATION: DESIGN

With this knowledge, we repeated the past experiment for 24 hours, but this time using our low number of copies of vector on this link pSB3K3.

At this time, the expression model for Ranaspumins production (explained in our model section ) had already predicted that the best production was obtained with 100 mM and 1000 mM of inducer, achieving a steady production since the first pair of hours after induction. With that information, we decided to use 100 mM and 1000 mM of inducer for our next set of experiments. The overall culture conditions were the same used in the first cycle: constant agitation at 200 rpm and 37°C).

BUILD

Based on the model predictions, we collected a 1mL sample for the culture induced with 100 mM and 1000 mM respectively, at 4, 6, 8, 10, 12, 20, 22, and 24 hours for their analysis on a SDS-PAGE gel, which is shown in the next image.


TEST

The concentrations of the expressed Ranspumin were determined by the pixel analysis program, ImageJ, which analyzes the difference between the density of pixels in the bands of an image from a SDS gel. The first step was to calibrate the program to a set of density standards, this can be done with the Calibrated Step Tablet provided in the ImageJ website (3)

Then, the program was tested out with an standar SDS gel, running different known concentrations (0.3, 0.1, 0.06, 0.03 and 0.01 mg/mL) of BSA according to Alonso and her collaborators (4). The final step was to analyze the SDS-PAGE gel and collect the results, for the complete analysis results you can see the complete file by clicking here .


LEARN

After analyzing the concentrations of the protein, we realized the expression was higher at 20, 22 and 24 hours, but started to be significant eight hours after induction. The obtained results are shown in the following table, where the highest concentrations are highlighted.

Rsn-2 induction 100 mM and 1000 mM
Condition Concentration (mg/mL) Peak
100 mM - 8 hours 0.1456 18.66
100 mM - 20 hours 0.0911 13.606
100 mM - 20 hours 0.3701 39.489
100 mM - 22 hours 0.0794 12.522
100 mM - 22 hours 0.4472 46.632
100 mM - 24 hours 0.0770 12.299
100 mM - 24 hours 0.6014 60.931


This behavior drastically differed from the one predicted by our model. For this reason, the gathered data was essential to feed and improve our model, making the proper adjustments that are detailedly described in our model section .

CYCLE 2, SECOND ITERATION: DESIGN

Since the difference between the concentrations with 100 and 1000 mM was huge, we carried out another experiment with different final concentrations of vanillic acid trying to find out if there was a more functional concentration of inducer. Hence, we made a second iteration of cycle two. The overall culture conditions were the same previously used: constant agitation at 200 rpm and 37°C).

BUILD

For this iteration, we used 100, 200, 400, 600, 800, and 1000 mM of our inducer, and only collected 1 mL sample at 20 and 24 hours for their analysis on a SDS-PAGE gel, which is shown in the next image.


TEST

After the analysis with ImageJ, we concluded that the best conditions for Rsn-2 production were 800 mM for 20 hours after the induction. The results are shown in the following table. The highest concentrations are highlighted.

Rsn-2 induction 100 mM to 1000 mM
Condition Concentration (mg/mL) Peak
100 mM - 20 hours 0.202 7.031
200 mM - 20 hours 0.0435 9.199
4000 mM - 20 hours 0.1171 16.017
600 mM - 20 hours 0.3929 41.6
800 mM - 20 hours 0.4253 44.608
1000 mM - 20 hours 0.2948 32.501
100 mM - 24 hours 0.0655 11.233
200 mM - 24 hours 0.0744 12.056
4000 mM - 24 hours 0.1148 16.138
600 mM - 24 hours 0.2081 24.46
800 mM - 24 hours 0.2099 24.624
1000 mM - 24 hours 0.2376 27.199


LEARN

In order to ensure our results were correct, we ran a SDS gel comparing Rsn-2 production induced with 800 mM of vanillic acid for 20 hours, against its production without adding any inducer. The results can be seen in the following image.

As observed, there is no expression of Rsn-2 without the addition of the inducer, meaning there is no basal expression.


THIRD CYCLE (Rsn-3 pSB3K3)

FIRST ITINERATION: DESIGN

Just as with Rsn-2, we used the first results of our model to shape our production experiments, hence, we decided to check out different concentrations of vanillic acid for 8 hours and analyze it with a SDS gel. The overall culture conditions were the same previously used: constant agitation at 200 rpm and 37°C).

BUILD

We collected a 1mL sample for the culture induced with 100, 400, 600, and 800 mM of vanillic acid 8 hours after induction, for their analysis on a SDS-PAGE gel, which is shown in the next image.


TEST

The production of Rsn-3 was determined using ImageJ. The obtained results are shown in the following table, where the highest values are highlighted.

Rsn 3
Condition Concentration (mg/mL) Peak
100 mM - 8 hours 0.3006 33.042
400 mM - 8 hours 0.4366 45.648
600 mM - 8 hours 0.3287 35.647
800 mM - 8 hours 0.5794 58.891
1000 mM - 8 hours 0.6014 60.932


LEARN

These experiments revealed that the best inducer concentration was 1000 mM. Due to this, we decided to make a second iteration of this cycle, with the aim of better understanding the functioning of our system’s production.

CYCLE 3, SECOND ITERATION: DESIGN

The next readjustment was to extend the experiment duration until 24 hours, since we wanted to find the best induction time with a final concentration of 1000 mM of vanillic acid. The overall culture conditions were the same previously used: constant agitation at 200 rpm and 37°C).

BUILD

We collected a 1mL sample for the culture induced with 1000 mM of vanillic acid at 4, 6, 8, 10, 12, 20, 22, and 24 hours after induction for their analysis on a SDS-PAGE gel, which is shown in the next image.


TEST

We observed that at 12 hours after induction.we had our highest protein production. The obtained results from ImageJ are shown in the following table. The highest values are highlighted.

Rsn 3
Condition Concentration (mg/mL) Peak
100 mM - 8 hours 0.1904 22.83
1000 mM - 10 hours 0.1471 18.807
1000 mM - 12 hours 0.5070 52.18
1000 mM - 20 hours 0.2381 27.238


LEARN

These continuous rounds of new information fed the expression model, which offered essential feedback suggesting the conditions in which the production would be more efficient. As it can be observed, this model was of great importance since it saved us effort and time. For reading the other side of this loop of information, visit our model section .

FOURTH CYCLE (Rsn-3-5 pSB3K3)

FIRST ITENARATION: DESIGN

Just as with Rsn-2 production, the expression model for Ranaspumins production had already predicted that the best production was obtained with 1000 mM of inducer, achieving a steady production since the first pair of hours after induction. Hence, we decided to repeat the 100 and 1000 mM experiment, but this time for Rsn-3-5 for 24 hours. The overall culture conditions were the same previously used: constant agitation at 200 rpm and 37°C.

BUILD

Based on the model predictions, we collected a 1mL sample for the culture induced with 100 mM and 1000 mM respectively, at 4, 6, 8, 10, 12, 20, 22, and 24 hours for their analysis on a SDS-PAGE gel, which is shown in the next image. In addition, we added a sample of Rsn-3 induced with 600 mM of vanillic acid at 24 hours (the same culture conditions previously mentioned) to visually compare the results of the production.


TEST

The obtained SDS-PAGE gel was analyzed to determine the concentrations using ImageJ. The obtained results are shown in the following table. The highest values are highlighted.

Rsn3-5 induction 100 mM / 1000
Condition Concentration (mg/mL) Peak
100 mM - 20 hours 0.2246 25.991
1000 mM - 20 hours 0.1215 16.425
100 mM - 22 hours 0.0896 13.474
1000 mM - 22 hours 0.0525 10.025
100 mM - 24 hours 0.1246 16.719
1000 mM - 24 hours 0.0812 12.691


LEARN

Once again, we observed a considerable difference between 100 and 1000 mM, but this time the concentration was higher at 100 mM of inducer and in fewer hours. With these results, we made a second iteration of this cycle, adjusting the parameters to better understand the expression.

CYCLE 4, SECOND ITERATION: DESIGN

We repeated the experiment with lower concentrations of inductor, using 8 hours as indicated on the first approach of our model. The overall culture conditions were the same previously used: constant agitation at 200 rpm and 37°C.

BUILD

We collected a 1mL sample for the culture induced with 10, 25, 50, and 75 mM of vanillic acid at 8 hours after induction for their analysis on a SDS-PAGE gel, which is shown in the next image.


TEST

We obtained the following concentrations of the expressed Ranaspumins using ImageJ. The highest values are highlighted.

Rsn 3-5
Condition Concentration (mg/mL) Peak
10 mM 0.0503 9.827
25 mM 0.0402 8.892
50 mM 0.0785 12.445
75 mM 0.0791 12.493


LEARN

The analysis with ImageJ revealed that the best inducer concentration was 75 mM. Once we determined the best inducer concentration, we decided to make a third iteration of the cycle to get to know the time of the highest protein production.

CYCLE 4, THIRD ITERATION: DESIGN

Finally, we extended the experiment for 24 hours. The overall culture conditions were the same previously used: constant agitation at 200 rpm and 37°C.

BUILD

We collected a 1mL sample for the culture induced with 75 mM of vanillic acid at 4, 6, 8, 10, 12, 20, 22, and 24 hours after induction for their analysis on a SDS-PAGE gel, which is shown in the next image.


TEST

The SDS gel was analyzed to determine the concentrations using ImageJ. The obtained results are shown in the following table. The highest values are highlighted.

Rsn 3-5
Condition Concentration (mg/mL) Peak
75 mM - 10 hours 0.0974 14.193
75 mM - 12 hours 0.2014 23.843
75 mM - 20 hours 0.1317 17.372
75 mM - 22 hours 0.1206 16.341
75 mM - 24 hours 0.1296 17.181


LEARN

Finally, we noticed that the major protein was 12 hours after the induction with vanillic acid. As occurred with Rsn-2, this data fed the already mentioned expression model of the Ranaspumins, which can be found in our model section .

WESTERN BLOTS

For validation, we realized a Western Blot analysis, transferring the SDS-PAGE gel to a nitrocellulose membrane. The obtained results are shown in the following images.


As shown in the figures, Rsn 3-5 is not appreciable in the Western Blot. This is probably due to the small amount of total protein production, since the production of the combined Rsn 3-5 is lower in comparison to Rsn-2 and 3, as previously shown in our production results.

PROTEIN PURIFICATIONS

Entering this stage of our process, we purificated every Ranaspumin using a Ni+ resin. As explained in the protocols document provided above. The SDS-PAGE gel is shown in the following images.


Another Western Blot was made to verify the correct purification of the proteins, shown in the next images. As last time, Rsn 3-5 is not visible, but a low intensity band in the normal induction and the purification, as seen in the image.


FOAM TESTS

Once we had our proteins production optimized, we carried out the tests to characterize the foaming properties of each one of our Ranaspumin proteins. The general objectives were to determine [1] the most effective method for foam creation, [2] the foamability and half-life of the sonicated proteins, and [3] the foamability and half-life of the different foam solutions. All the experiments were carried out in environmental conditions (25°C), the complete methods and results are explained below.

Taking into consideration the evaluation of foaming compounds developed by Petkova (5), in which she applied agitation to corning tubes; we Designed our next set of experiments. Hence, we tested this method (hand shaking for 30 seconds) against the pressure release using a syringe (control). In addition, we also took into account the times for the study of surfactants and stabilizers. We used 24 hours given the natural Ranaspumins durability reported by Mackenzie (6) and the time used in literature (5). Since this time we wanted to test the effect of the surfactants against a solution with no surfactants on it, we used water as the negative control.

DETERMINATION OF THE BEST FOAMING METHOD

DESIGN
For this experiment, we used 15 mL corning tubes containing 5 mL of each of our sonicated proteins from the culture media with the optimized conditions previously reported. All the test conditions followed the general conditions explained at the beginning of this section. The aim of this experiment was to prove if the agitation (used for mechanical firefighting foams) would be effective with our components.

BUILD

We measured the volume of foam using the mL scale of the corning tube at different times and recorded it.

TEST

In the following image, we can observe the initial volume (mL) of foam and the initial volume (mL) of liquid containing our proteins.


After analyzing the results it was found that, on average, the stirring method generates 0.74 mL more foam per mL of initial solution compared to using the syringe. This is shown most strongly in the foams of Rsn-2, which generates 3.6 times more foam with agitation than syringe.

LEARN

Through our results analysis, the agitation proved to be a much more effective method than the use of the syringe for the creation of foam; with this result we proved Ranaspumin proteins must be efficient for a mechanical firefighting foam, since agitation is the best foaming method. In addition, we realized the vital importance of standardizing the concentrations of the concentrates. Thus, to study the foamability and half-life, the initial concentrations will be controlled during the following studies.

CHARACTERIZATION OF THE PROTEINS PHYSICOCHEMICAL FEATURES

DESIGN

Considering our previous conclusions, we took the highest concentrations, obtained in our characterization of the Ranaspumins induction experiments explained above. They are as follows: 0.4253 mg/mL of Rsn-2, 0.5070 mg/mL of Rsn-3, and 0.2014 mg/mL of Rsn-3-5. The correct ratio to have the same amount of protein is shown in the table above, these amounts of sonicate will be diluted to perform the foamability and half-life tests at the same protein concentrations.

Foam Solution Rsn-2 Rsn-3 Rsn-3-5
mL 2.37 mL (1.01 mg) 1.99 mL (1.01 mg) 5.0 mL (1.01 mg)


BUILD

The experiment was performed with the same conditions explained in the past experiment, with the exception of the specific concentrations shown in the table above (1.01 mg/ 5mL) = 0.202 mg/mL for Rsn-2, Rsn-3, and Rsn-3-5. Here, we used SDS as a positive control, in order to compare our foam with a well-known surfactant.

TEST

In the following image, we can observe the initial volume (mL) of foam and the initial volume (mL) of liquid containing our proteins.


As expected, water did not create foam, while SDS produced a foam with a volume of 1.9 mL per inicial volume. Of all the Ranaspumins, the Rsn-3-5 generated the greatest amount of foam, specifically 0.9 mL per mL of initial sonicated volume. Followed by Rsn-2 and Rsn-3, with 0.8 times the initial volume in foam. Then, it can be observed that Rsn-3 has a very similar foamability to Rsn-2 under the same concentrations.

In the next image, we show the volume of foam in mL generated by water, Rsn-2, Rsn-3, Rsn-3-5, and SDS at 12 hours.


Regarding the durability, the Rsn-2 sonicated foam dropped after 510 minutes (8.5 hours), while the one of Rsn-3 sonicated foam passed 720 minutes (12 hours) of experimentation with more than 50% of initial volume. On the other hand, Rsn-3-5 sonicate foam passed 540 minutes (9 hours) of experimentation still at 50%; Finally, positive control SDS lasted 720 minutes (12 hours) with 74%. This supports our hypothesis since Ranaspumins 3, 4, 5 are known to be foam stabilizers based on their lectins’ characteristics making the foam more durable.

LEARN

In terms of half-life, Rsn-2 and Rsn-3-5 showed the lowest half-life of 510 minutes (8.5 hours). On the contrary, Rsn-3 presented the longest half-life, passing 12 hours with 50% of the initial foam volume.

CHARACTERIZATION OF THE FOAMS PHYSICOCHEMICAL FEATURES

DESIGN

In order to have a final formulation of our foam, based on the features previously describe, we Designed different foam formulations based on the proportion of each of the elements (Rsn-2: 0.4253 mg/mL, Rsn-3: 0.5070 mg/mL, Rsn-3-5: 0.2014 mg/mL). Hence, we made the following formulations to make the proper tests and determine the best final foam.

Solution 1: 1/3 sonicated of Rsn-2 culture and 1/3 of sonicated of Rsn-3, and 1/3 of sonicated of Rsn-3-5 culture:

0.4253x= 0.5070y=0.2014z
x+y+z=5
Rsn-2=1.26 mL Rsn-3 =1.06 mL Rsn- 3-5=2.67 mL

Solution 2: 2/4 sonicated from Rsn-2 culture and 1/4 sonicated from Rsn-3 culture and 1/4 sonicated from Rsn-3-5 culture:

0.4253x= 0.5070y+0.2014z
0.5070y=0.2014z
x+y+z=5
Rsn-2=2.02 mL Rsn-3 =0.85 mL Rsn- 3-5=2.13 mL

Solution 3: 3/5 sonicated from culture Rsn-2 and 1/5 sonicated from culture Rsn-3 and 1/5 sonicated from Rsn- 3-5 culture:

0.4253x= 1.667 0.5070y+0.2014z
0.5070y=0.2014z
x+y+z=5
Rsn-2=2.59 mL Rsn-3 =0.68 mL Rsn- 3-5=1.72 mL

Solution 4: SDS Solution with the same concentration as protein concentration solution 3 (1.79 mg).
TEST

In the following image, we can observe the initial volume (mL) of foam and the initial volume (mL) of liquid containing our proteins. Of all the combinations, the Solution 3 generated the greatest amount of foam, with 0.9 times the initial sonicated volume. This was followed by Solution 1 and 2, with 0.8 times the initial volume in foam. As in all the past experiments, water did not create foam. Meanwhile, SDS formed a foam with a volume of 1.9 times.


As it can be observed in the image below, the Solution 1 foam dropped after 630 minutes (10.5 hours), the Solution 2 sonicated foam 720 minutes (12 hours), and finally, the Solution 3 foam passed 600 minutes (10 hours) of experimentation still at 50%. Last, the positive control SDS lasted 720 minutes (12 hours) with 74% of the initial volume.


Since Rsn-3-5 are known to be foam stabilizers based on their lectins’ characteristics, it can be explained its effect on the foam extended durability. In fact, the most durable solution is the second one, in which Rsn 3-5 are found to a greater extent (Rns-2 2/4, Rsn-3: 1/4, Rsn 3-5: 1/4. However, it shows less foamability than the foam with the higher amount of Rsn-2 (Solution 3, Rsn-2: 2/3, Rsn-3: 1/6, Rsn 3-5: 1/6).

Learn

With this experiment, Solution 3 was chosen due its large foamability and lasting half-life. Then, our final foam composition was Rsn-2 at a concentration of 60% (vol/vol), Rsn-3 at a concentration of 20%, and Rsn 3-5 at 20% of the concentrate. Here, even though Rsn-2 was not the most efficient behavior (compared to Rsn-3 and Rsn-3-5), we noticed that it has to be the most abundant protein in the final foam formulation. If you want to learn more about the tests we made with the foam please visit ourproof of concept section. This is probably due to interactions between the proteins, but further research is needed to prove this hypothesis.

GENERATING DATA TO FEED OUR MODEL

Modeling is a very important part of the development of a project. However, in some cases they require experimental data in order to work properly. Next, we present some of the experimental work we developed to feed our models; the detailed information of how we used the experimental outcomes is explained in our model section .

MODELING A FED BATCH BIOREACTOR FOR THE PRODUCTION OF RSN 2-5 USING E. coli

For this simulation, we needed a growth curve of our E. coli Top 10 cultures with the production genes inserted. The curve was determined experimentally reading the absorbance at 600 nM of different samples for 24 hours. Then, we obtained an average of the three cultures. With this, the model was fed, allowing us to theoretically up-scale the production of our proteins. For more information about this model, please visit our model section .

OPTIMIZATION OF CULTURE CONDITIONS FOR RSN PRODUCTION IN E. coli USING SURFACE RESPONSE METHODOLOGY

On the other hand, we also developed a model that would allow us to