Team:TecCEM/Results

Cell Gif

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?

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

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

  1. First stage : E. coli transformation
  2. Second Stage: Protein production
  3. Third Stage: Test in cellular line

First stage

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 posterious transformations into E. coli. We had several transformation attempts (Figure 1), which, mostly of the times no adequate migrations patterns could be achieved. We carried on with bacterial transformation and protein production, as we thought that bands of ligation could not be saw given the agarose sensibility, surprisingly bacterial colonies (either E. coli BL21(DE3) and DH5 alpha ) could be saw (Figure 2) in selective medium but no constant protein production could be achieved (Go to second stage for more information). After many failed attempts and a lot of time invested, we suspected that maybe ligations were falling because of the absence of close to the end DNA cleavage nucleotides (needed for restriction cutting). For that primers were designed to tackle this problems using iGEM prefix and suffix sequences as template for amplification. We PCR amplified BBLEP (Figure 3) and ligate it into psB1C3 (Figure 4), 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 5), sadly because of time circumstances and embedded DNA synthesize problems, BBCOL and BBTNC could not be tested with this methodology. Future plans include making 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.
Clave 3
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)

Tenascin confirmation (protein level)

Clave A
Figure 5.
  • 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