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 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 second stage for 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)

Second stage

Protein production

After doing transformation into E. coli BL21 (DE3) we started the production and purification of our protein of interests. This stage 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.

• 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: In the gel there is 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