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.

• 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)

• 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)

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)

• 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.

Tenascin and leptin confirmation (protein level)

• 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.

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.