Results

Overview

To accomplish our project objective several recombinant proteins must be produced in order to analyze their effects on a co culture of fibroblasts (L929) and mesenchymal cells after an in vitro burn assay. To evaluate the efficiency of the treatment, a measurement of the proliferation rate was performed using LDH (lactate dehydrogenase) as an analysis metabolite. Given that our treatment involves the usage of a growth factor, the rate at which this protein is released into the medium is critical to avoid adverse effects in the cellular line (such as cancer), for that reason a nano encapsulation with chitosan was also performed to control the rate at which our growth factor is released.

Summary

What did we accomplish?

Nanoencapsulation in chitosan of leptin, BSA and RFP.
Realization of in vitro burn assay.
Realization of leptin proliferation essay.
Obtaining parts with adequate enzyme recognition sites.
Protein production of leptin and tenascin C (More validations are needed).
Co-culture of fibroblast and mesenchymal cells.
Cellular growth in TaCO-BOB hardware.

What happened?

We started ligating parts, no bands of expected size were observed but we attributed this fact to low sensitivity of agarose gels, so we proceeded with transformation and protein induction. Nevertheless, we realized that consistent results were not achieved (bad protein migration rates, sometimes no induction band could be observed). [Unfortunately, we invested a lot of time on this period]
Then we started thinking that our parts did not had the necessary extra nucleotides for proper cleavage of restriction enzymes and what we were truly observing was the effect of star activity and inappropriate ligation, resulting in non specific plasmids which did not contained our whole part but conferred the bacteria antibiotic resistance.

How did we solved it?

We designed primers that incorporate the necessary nucleotides into our parts. Thus, we started having proper migrations of project parts and posterious transformations and protein production of BBLEP.

How should we improve it?

By including better protein reporters for the screening of transformed, functional bacteria colonies.

Our proyect is divided into three sections which are the following:

Phase 1: E. coli transformation
Phase 2: Protein production
Phase 3: Test in cellular line

Phase 1 E. coli transformation

Once our IDT synthesis arrived, we set the objective of ligating the composite parts of tenascin (BBa_K2719005) [also called BBTNC], leptin (BBa_K2719009) [also called BBLEP] and collagen (BBa_K2719007) [also called BBCOL], into an RFC10 compatible psB1C3 iGEM plasmid for future transformations into E. coli. We had several transformation attempts (Figure 1), most of the times there are no adequate migrations patterns achieved. We carried on with bacterial transformation and protein production, as we thought that bands of ligation could not be seen given the agarose sensibility, surprisingly bacterial colonies (either E. coli BL21(DE3) and DH5 alpha ) were seen (Figure 2) in selective medium but no constant protein production could be achieved (Go to phase 2for more information). After many failed attempts and a lot of time invested, we suspected that maybe ligations were failing due to the absence of “close to the end DNA cleavage nucleotides” (needed for restriction cutting). To solve the problem previously mentioned, primers were designed to tackle this problems using iGEM prefix and suffix sequences as template for amplification. We amplified BBLEP (Figure 3) and ligated it into psB1C3 finally obtaining the expected sizes at each step. Thus with this methodology we could resolve the difficulties for BBLEP and achieving a transformation in BL21(DE3) (Figure 4), due to timewise limitations and the embedded DNA synthesize problems, BBCOL and BBTNC could not be tested with this methodology. Future plans include applying that methodology to those parts and transforming into respective bacteria strains.

Clave 1 — Figure 1. First attempt of ligation of IDT parts into a psB1C3 plasmid

Experiment: Electrophoresis.
Matrix: Agarose (0.85%).
Conditions: 100 volts, 60 minutes in TBE buffer.
The samples were charged in the following order:
1. 2-log DNA ladder (6uL)
2. BBLEP 2739bp (3uL + 3uL of loading buffer)
3. BBCOL 3566bp (3 uL + 3uL of loading buffer)
4. BBTNC 3196bp (3 uL + 3uL of loading buffer)
5. BBTNC 3196bp (3 uL + 3uL of loading buffer)
6. 2-log DNA ladder (6uL)
7. 1kb DNA ladder (6uL)

Clave 2 — Figure 2. Culture plates containing transformed *E. coli* .1) *E. coli* DH5 alpha containing BBCOL 2) *E. coli* DH5 alpha containing BBLEP 3) *E. coli* DH5 alpha containing BBTCD.

Figure 3. BBLEP amplicon obtained after PCR with designed primers

Experiment: Electrophoresis
Matrix: Agarose (0.85%)
Conditions: 100 volts, 60 minutes in TBE buffer
The samples were charged in the following order:
1. 2-log DNA ladder (6uL)
2. BBLEP PCR amplicon (3 uL + 3uL of loading buffer)

Clave 4 — Figure 4. Culture plates containing transformed *E. coli* BL21(DE3) with BBLEP.

Phase 2 Protein production

After doing transformation into E. coli BL21 (DE3) we started the production and purification of our protein of interests. This phase was a key factor for the demonstration of the functionality of our project. First, we obtained polyacrylamide gels that had clear signs of maximum induction of our proteins at 5 hours after IPTG induction. The results that we obtained were inconsistent between several repetitions of the induction.

After that observation several hypotheses were formulated in order to explain those events, the first one was that the synthesis sequence had several errors, which made the peptide product unstable, and an easy target for degradation. This hypothesis was mainly supported by the apparition of the protein at 5h after induction, but its complete disappearance after 16 hours, specifically TCD that needs a protein fusion to improve its stability and reduce the rate of degradation.

The second hypothesis was that the used strain (E. coli BL21 (DE3)) was mislabeled, therefore the transformation and induction were on strains unable to produce RNApol T7. This idea was discarded after several experiments were made on fresh strains, obtaining the same inconsistent results. Even though the results continued to be inconsistent, with the fresh strains some improvement was made. Tenascin was observed for the first time on SDS-PAGE after 5h of induction (see figure 5). Also a Dot Blot was performed in order to evaluate the presence of Tenascin and Leptin using the His-TAG (see figure 7).

The third and last hypothesis was that an error on the synthesis meant that there were not enough nucleotides for allowing the restriction enzymes (EcoRI and PstI) to work properly, reducing ligation efficiency. In order to test this hypothesis new primers were developed, those primers after amplification with PCR, added extra nucleotides to the prefix and suffix o the parts promoting the properly work of the enzymes.

Finally, since E.coli BL21 (DE3) has a protein that migrates at the same levels [1] as those expected for leptin, a Western Blot was performed in order to get more specific results. Using an anti-His antibodies we were able to determined the presence and the production of the leptin. In order to do this, a inoculum of transformedE.coli BL21 was induced at a concentration of 1mM IPTG. The samples were lizated utilizing RIPA buffer and were loaded on an gradient 5%-20% BioRad SDS-PAGE. This was transferred to a nitrocellulose membrane and incubated with anti-His antibodies. The results were positive, obtaining bands (where the antibody binds) at a size close to 17 kDa, similar to the expected leptin molecular weight (see figure 8). So the production of leptin is proposed, but further experimentation is required to standardize its production.

Tenascin confirmation (protein level)

Clave A — Figure 5. Analysis of BBLEP and BBTCD before IPTG induction

Experiment: SDS-PAGE
Matrix: Polyacrylamide (15%)
Conditions: 1080 volts for 20 minutes, 120 volts 60 minutes.
Objective: Determine the presence of protein from BBLEP (BBa_K2719009) and BBTCD (BBa_K2719005) in protein producer strain E. coli BL21 (DE3)
Description: Crude lysate (30 uL + 10 uL laemmli buffer) coming from transformed BL21 (DE3) strains and induced with 1 mM IPTG for different hours at 37 Celsius degrees were loaded onto polyacrylamide gel and runned with SDS-Tris-Gly buffer
The samples were charged in the following order:
1. BBTCD V2 (At 16 hours of induction) [BBa_K2719005]
2. BBTCD V1 (At 16 hours of induction) [BBa_K2719005]
3. BBLEP V2 (At 16 hours of induction) [BBa_K2719009]
4. BBLEP V3 (At 16 hours of induction) [BBa_K2719009]
5. BBTCD V1 (At 16 hours of induction) [BBa_K2719005]
6. BBLEP V1 (At 16 hours of induction) [BBa_K2719009]
7. BBTCD V2 (At 5 hours of induction) [BBa_K2719005]
8. BBTCD V1 (At 5 hours of induction) [BBa_K2719005]
9. BBLEP V2 (At 5 hours of induction) [BBa_K2719009]
10. Protein Ladder
Analysis: The gel has a constant band pattern among lanes, which represents the native proteins of BL21 (DE3) strain. On the other hand, a single band is highly ovestanding which can be seen in lane 7, here a single pattern band between 25 and 37 kDa correspond to a possible apparition of BBTCD protein, which has a theoretical weight of 35.1 kDa, the band stand as very likely candidate as its form and luminosity correspond to a common protein band derived from an IPTG expression, nevertheless, at 15 hours the same part (lane 1) shows no sign of the protein, this can be explained as the lost of function of IPTG given by continuous exposure to high temperatures and consequently loss of protein production possibility and gradual degradation of existing protein. For BBLEP no sign of protein production could be recorded. More analysis to BBTCD must be done to corroborate the existence of protein, and a restructure of troubleshooting for BBLEP must be done to create better results of production..

Selection of Tenascin producer strain

Experiment: SDS-PAGE
Matrix: Polyacrylamide (20%)
Conditions: 80 volts for 20 minutes, 120 volts 60 minutes.
Objective: Determine the E. coli BL21 (DE3) strain that produce the protein BBTCD (BBa_K2719005).
Description: Crude lysate (30 uL + 10 uL laemmli buffer) coming from transformed BL21 (DE3) strains and induced with 1 mM IPTG for different hours at 37 Celsius degrees were loaded onto polyacrylamide gel and runned with SDS-Tris-Gly buffer.
The samples were charged in the following order:
1. BBTCD V1 (At 5 hours of induction) [BBa_K2719005]
2. BBTCD V3 (At 5 hours of induction) [BBa_K2719005]
3. BBTCD V2 (At 5 hours of induction) [BBa_K2719009]
4. Negative control E.coli BL21 (DE3) without transformation (At 5 hours of induction)
5. Protein Ladder
Analysis:Analysis: A single band is highly overstanding which can be seen in lane 3, here a single band of 25 kDa that confirm the possible presence of BBTCD protein in the BBTCD V2 strain, furthermore there is an inconsistency in this line because the theoretical weight of TCD is 35.1 kDa. Obtaining an unexpected molecular weight for BBTCD as a fusion protein, made us suggested that the protein sequence is incomplete. This aceveración can explain the reason why there is no presence of BBTCD at 16 hours of induction. The GST as a fusion protein helps for the stability of the entire protein. Its absence difficult to have the protein in the stable form and it is possibly degraded.

Clave B — Figure 6. Analysis of BBTCD in different *E. coli* BL21 strains

Tenascin and leptin confirmation (protein level)

Clave C — Figure 7. Dot Blot analysis of BBTCD and BBLEP

Experiment: Dot blot
Matrix: Nitrocellulose membrane
Conditions: Incubation with Anti His tag antibody (dilution 1:1000) for 1 hour and revelation by Dye Green transilluminator protocol.
Objective: Determine the presence of protein from BBLEP (BBa_K2719009) and BBTCD (BBa_K2719005) in protein producer strain E. coli BL21 (DE3)
Description: Crude lysate coming from transformed BL21 (DE3) strains and induced with 1 mM IPTG for 5 hours at 37 Celsius degrees were loaded onto nitrocellulose membrane (3 uL) and incubated with Anti His tag antibody coupled with a fluorochrome reporter.
The samples were charged in the following order:
1. Negative control E.coli BL21 (DE3) without transformation (At 3 hours of induction)
2. Negative control E. coli BL21 (DE3) without transformation (At 5 hours of induction)
3. BBTCD V2(At 3 hours of induction) [BBa_K2719005]
4. BBTCD V2(At 5 hours of induction) [BBa_K2719005]
5. BBLEP V1 (At 3 hours of induction) [BBa_K2719009]
6. BBLEP V1 (At 5 hours of induction) [BBa_K2719009]
7. BBLEP V2 (At 3 hours of induction) [BBa_K2719009]
8. BBLEP V2 (At 5 hours of induction) [BBa_K2719009]
9. Positive control
10. Nothing
Analysis: After the inconsistent results obtained in the polyacrylamide gels, a Dot Blot was executed. Observation of colored points in positions 3, 5 an 7 gives a positive result in presence of recombinant proteins which contain a histidine tag, in positions 4, 6 and 8 an even more colored point is detected, this confirm that the production of protein increases significantly after 5 hours of induction. On the other hand positions 1 and 2, which accounts for negative control, resolves that E. coli BL21 (DE3) does not produces endogenous native proteins with a high content of histidine tags that could be misunderstood as our protein, this event partially eliminates false positive results. Meanwhile position 9, which account for positive control, shows no coloured point, this effect must be due to the high degree of dilution that we applied to the positive control (1:1000), also there is no visualization of the mark that normally happens when the sample dries, thus we can conclude that our positive control, which was a recombinant protein from E coli, was not in optimal state. This result complicates the final decision about our protein production, for that more dot blots or even western blots must be applied for a better confirmation of results.

Experiment: Western blot
Matrix: Nitrocellulose membrane
Conditions: Incubation with Anti His tag antibody (dilution 1:1000) for overnight.
Objective: Determine the presence of protein from newly ligated BBLEP (BBa_K2719009) in protein producer strain E. coliBL21 (DE3).
Description: CCrude lysate coming from transformed BL21 (DE3) strains and induced with 0.5 mM IPTG for 5 hours at 37 Celsius degrees were loaded onto nitrocellulose membrane (3 uL) and incubated with Anti His tag antibody coupled with a fluorochrome reporter. At left SDS-PAGE 15% gel revealed with Coomassie blue for 30 minutes, at right western blot.
The samples were charged in the following order:
1. Molecular weight ladder Spectra.
2. BBLEP (At 0 hours of induction)
3. BBLEP (At 2 hours of induction)
4. BBLEP (At 4 hours of induction)
5. BBLEP (At 6 hours of induction)
Analysis:As is observed on the SDS-PAGE, is almost impossible to distinguish between the native E.coli BL21 (DE3) protein and leptin, as both proteins migrate in a similar way, therefore both bands render undistinguishable. To overcome this problem, a Western Blot was performed using an anti-His antibody, taking advantage that leptin contains a 6xHis Tag and the native protein does not, so the antibody will only bind to leptin. The results after the incubation with the antibodies determined not only the presence of a peptide of around 17 kDa with an histidine Tag, presumably leptin. But also as the width of the band increased as the induction time increased (for times 0, 2h, 4h and 6h), thus suggesting a successful IPTG induction of the part.

As is shown on the Western Blot (see figure 8) recombinant leptin was successfully produced by our parts in E.coli BL21 (DE3), but the degree of purity obtained was not sufficient for its usage on a cell line. But, as a proof of concept was necessary to demonstrate the effect of recombinant leptin on cell proliferation in burn damaged tissue. Then, we decided to use high degree of purity commercially available leptin for our assays on cell lines. In this way, the functionabillity of our project is demonstrated on a real experimental manner, minimizing the effect of external variables (such as contamination from undesired components).

Phase 3: Nanoencapsulation and test in cellular line

As a delivery vehicle was needed protein-loaded chitosan nanoparticles were prepared by ionic gelation method. For a correct standardization of the encapsulation protocol, we used BSA as protein. BSA is often employed for concentration measurements in protein assays, including encapsulation efficiency and protein release in a solution of loaded nanoparticles.

Chitosan nanoparticles were prepared with a pH of 5.0 since BSA has an isoelectric point of 4.7 at 25 °C with TPP polyanion under mild conditions following our standardized protocol.

What we expected:

We wanted to create an efficient drug delivery method that would regulate leptin liberation into the cells as an uncontrolled amount of leptin can cause a swelling reaction. By standardizing the nanoparticle particle size, particle count, encapsulation efficiency, and protein release behavior in a physiological pH solution we expected to accurately predict a cell proliferation increase in treated cell lines with nanoparticles against non-treated.

What we achieved: Characterization of nanoparticles

For the encapsulation of BSA at pH at 5.0 a particle analysis in NanoSight NS300 to obtain the particle size distribution was performed. A dot graph for a triplicate analysis is presented below.

Particle size and particle count

Particles were analyzed in NanoSight NS300, from which we obtained 127.8 nm as mean particle size with a broad standard deviation of 90.1 nm. NanoSight NS300 displays a video when analyzing a sample volume of nanoparticle solution, which we include here.

Figure 9. RFP loaded nanoparticles graphs of concentration (particles/mL) vs Size (nm) obtained from NanoSight NS300

Analysis:

Some chitosan nanoparticles displayed a bigger diameter, this might be a result from the swelling of chitosan layer and aggregation of single particles while dispersed in water, causing a detection of several bigger particles, shifting the mean value and standard deviation. Transmission electron microscopy (TEM) was used as a second method of confirmation, in which nanoparticle morphology was observed. We think this difference is caused by the sample treatment required for TEM, that enables adequate particle distribution to observe them individually. A mean size by TEM was determined to be 27.7 nm.

Encapsulation efficiency

A Bradford assay was performed to determine the protein encapsulation percentage in order to evaluate the efficiency of the protocol used to form loaded chitosan nanoparticles. The recovery of protein from the encapsulation procedure was 42% at the stated conditions.

Protein release behavior

We established the relation between the amount of protein released by nanoparticles with respect to time, measuring the protein concentration at 0, 2, 4, 12, 18 and 24 hrs. Nanoparticles were incubated at 37 °C and 100 rpm suspended in PBS pH 7.4 to estimate how they would behave in a physiological medium. After 24 hours almost 50% of the protein was released.

Leptin loaded chitosan nanoparticles action in cell lines

As the main target of this project was to prove protein release from a chitosan encapsulation that would increase cell proliferation in a cell line, we encapsulated leptin under the same conditions. Afterward, leptin-chitosan nanoparticles were tested in L929 fibroblasts cell line which was damaged with a hot solution at different degrees. For every degree of damage, a well was supplied with 3.4 ng/mL of leptin growth factor. Results analyzed after 72 h are presented below. Commercial leptin growth factor effect on burned L929 fibroblasts is demonstrated through the performance of this experiment.

Cell line burn assay standardization

Burn injuries represent a serious issue that involves pathophysiological changes, it is important to develop new mechanisms to test the viability of improvements or new treatments. For that reason,we decided to perform an assay which consisted of analysing cell proliferation after submitting L-929 cell line to a burning procedure. This allowed us to monitor if the evaluation of our treatment through this mechanism was feasible.

After setting different exposure times (15, 30, 45 and 60 seconds) to which each well would be exposed to HBSS (Hank solution) at 60ºC, we registered the results comparing the damage taken by the cell culture in each of the wells. We can conclude that the longer the cells are exposed to the solution, the more damage they take and the less cellular density is observed. This analysis allowed us to proceed with the evaluation of TecTissue performing another burn assay to apply later the treatment we designed. The importance of the standardization lies in the fact that it establishes the way in which we will perform the following assays to determine the efficiency of our project and set the basis for a more specific complex burn model.

In this in vitro burn model, it is possible to observe the increasing cell injury and significant changes in cell viability and morphology in relation to the time they were exposed to the hot HBSS.

In conclusion, the exposure of the culture to hot HBSS caused visible damage to the cells, causing possible cell necrosis and cell detachment of cell culture flasks. The different times employed are directly proportional to the harm taken by the culture and therefore the number of cells is visibly reduced. This assay allows researchers to perform in vitro analysis of burn process and the nature of the procedure makes it a feasible and reproductive alternative to study the cellular and molecular alterations throughout the burn process. In perspective, we could measure necrosis, cell viability, LDH, among others.

Burn assay with treatment

Once the burn assay was standardized, it was time to prove the efficiency of TecTissue. For that reason, we performed another burn assay with the times stated before but applying the hot HBSS to a co-culture of L-929 fibroblasts and human mesenchymal stem cells. Since our project is based on the combination of several factors, we decided to test the activity of each throughout the regeneration of the cultures.The compatibility of both cell lines allowed us to perform the procedure without any complication. However, due to the importance of the procedure, an MTT assay was performed additionally to ensure the accuracy of the results obtained and to legitimate the conclusions reached after the assay. The following pictures show the amount of cells depending on three factors:

Time of exposure to hot HBSS
Treatment given after burning the co-culture
- Chitosan nanoparticles with leptin encapsulated
- Empty chitosan nanoparticles
Co-culture of fibroblasts and mesenchymal cells

According to the figures shown, the damage taken by the cells is visible related to the amount of time they were exposed to the hot solution; however, in order to determine at a molecular level the viability of the cells remaining, we performed an MTT assay with the purpose of relating the proliferation of the cells with the efficiency of the treatment.

This figure compares the proliferation of the co-culture when one of the samples is treated with all the elements that form part of TecTissue (collagen, heparan, leptin nanoparticles) and the other with empty nanoparticles. The graph shows that the dosified release of leptin increases the proliferation and regeneration of the cells while the ones that received the treatment without the leptin did not have the same response. This behaviour is due to the activity of the leptin, which is slowly released into the matrix allowing cells to absorb it and enhance their metabolic activity to proliferate. Nevertheless, we can observe that empty chitosan nanoparticles may cause some response.

After analysing the results obtained from the MTT assay, we confirmed our hypothesis and observed that cells treated with TecTissue were indeed metabolic enhanced and confluent compared to the ones that were not treated. Some aspects to consider are that even though the leptin, heparan, and collagen were studied, none of them were evaluated separately, which means that further analysis should be performed to determine the activity of each element within the treatment. As stated in the corresponding sections, chitosan, heparan and collagen each play a specific role in our project and the combination of them represent at first glance, a good opportunity to develop new treatments for burn injuries. Even though we demonstrated the efficiency of TecTissue, it is important to mention that within 72 hours, the confluence of both conditions (charged and empty NP), present no significant differences. This means that the effects of our treatment are more visible within the first 48 hours. Since confluence of both wells were similar, we can say that TecTissue is a feasible treatment but within the short term. Further analysis should be made to determine whether it is possible to increase the efficiency of the treatment considering the areas of opportunity identified after the assay.

Another thing that needs to be sorted out, is the relation between the co-culture and the damage induced, with the presence or absence of leptin within the nanoparticles because the results obtained with the microscope suggest that the chitosan alone could plays a key role in the regeneration and proliferation of the fibroblasts.

Furthermore, it is necessary to determine the efficiency of the project, measuring metabolites such as Lactate Dehydrogenase (LDH), proliferation cytokines like tissue growth factor beta, which is known to be released by the mesenchymal stem cells due to the action of leptin.

The amount of cells that can be seen in each well of the plate are directly related to the time it was exposed to the hot HBSS, the treatment employed and the time it was left incubated afterwards. For that reason, it is important to describe the role of each element of the treatment and its influence in the regeneration of the cells: Collagen and heparan as the scaffold, leptin as the growth factor and chitosan as the vehicle that regulated the release of the drug. The combination of all these, allowed cells to react positively and enhance the proliferation, increasing its rate and potentiating the healing process.

Cell culture in BOB

Part of the evaluation of TecTissue consisted in testing the feasibility of our hardware, which was designed with the purpose of automatizing 2D/3D cell cultures. In order to evaluate if BOB is useful, we decided to perform a co-culture within it and analyse if cells were able to adhere to the PDMS surface and proliferate. The following pictures display how cells behaved when cultivated in BOB. To achieve our goal, we had to adequate the hardware with the molecules necessary for the correct adherence of the cells. In other words, we tested several combinations of the elements that comprise TecTissue. For example, a BOB with a scaffold made out of PDMS supplemented with collagen and heparan. The same study was performed with other combinations of said elements and removing the PDMS. After incubating for at least one day, we photo documented and recorded the results observed in a microscope employing fluorescence to ensure cells were correctly adhered to the synthetic matrix.

The results demonstrate that porous-collagen-based scaffolds membrane are effective for cell culture and tissue engineering. The images obtained from the epifluorescence microscope with a mitochondrial dye allow us to conclude that employing these elements it is possible to perform a co-culture. This is supported by the immunofluorescence assay that allowed us to see the correct attachment of the cells. Further analysis should be performed to enhance the efficiency of the PDMS and increase the cellular adhesion.

Team:TecCEM/Results