Overview

The main aim for our project was to produce recombinant proteins that would compose our scaffold to improve cell proliferation. Throughout our literary research, we came across scaffolds, which were the ideal support that would improve cell-cell interaction, cell migration, proliferation and differentiation. We approached the scaffold design with three different proteins, each with properties to achieve better tissue regeneration and could interact with each other. This meant we needed a way to produce protein overexpression in E. coli vectors.

In this section the description of the constructs and background behind the constructs is explained.

Collagen Biobrick

1. A collagen-like synthetic gene comprising heparin binding site of collagen type V (HepV) was employed. The synthetic gene for COL5A1 was optimized for codon usage in E. coli Rosetta 2 (DE3), whereas illegal sequences were removed. 2. The nucleotide sequence of collagen is optimized for E. coli Rosetta 2 (DE3) and cloned into the backbone pSB1C3 that consists in a chloramphenicol resistant gene using EcoRI and PstI restriction sites, with an inducible T7-lac promoter for the construction of the BioBrick. The T7-lac promoter is extracted from plasmid pET-DEST42. 3. A nucleotide sequence encoding Red Fluorescent Protein (RFP) was retrieved from iGEM database (BBa_K1323009) and cloned into pSB1C3 under an Anderson constitutive promoter (BBa_K823005) using a RFC[10] with restriction enzymes: EcoRI and PstI and used a double TAATAA stop codon. The resulting plasmid is referred to as pSB1C3-RFP-COLlikeHepV. To determine whether the sequence of the resulting gene would produce the same protein a simulation of the protein was done with I-Tasser.

Tenascin Biobrick

1. The TNC domain of fibronectin type III-like repeats (TNCIII), specifically the subdomain TNCIII5 has the responsibility of the binding with heparin. This binding between heparin and tenascin is involved in affinity with many growth factors, specifically with Fibroblast Growth Factor (FGF). 2. The nucleotide sequence of TNCIII5 is optimized for E. coli BL21 DE3 and cloned into the backbone (pSB1C3) using EcoRI and PstI restriction sites, with an inducible T7-lac promoter and rrnB-T1 terminator using RFC[10]. The domain was expressed by bacterial expression as GST fusion protein and use C-terminal HIS tag. The fusion protein GST was used for the stability of the domain. 3. A polyproline linker was used between the TCDIII5 domain and GST in order to conserve the three dimensional structure of the full protein and then a protein simulation was done using I-Tasser simulator.

Leptin Biobrick

1. Leptin is a growth factor and is the main protein that helps in this project for skin regeneration. This is going to be encapsulated by chitosan and binded to the scaffold composed by Tenascin, collagen and heparin. Leptin protein is the product of the ob gene and it is found in adipocytes cells. 2. The coding sequence is optimized for Escherichia coli BL21 (DE3) and cloned in a pSB1C3 plasmid. To get the cloned sequence is necessary the implementation of EcoRI and PstI restriction sites, that they are located in the prefix and suffix, respectively, previously sitetyzed for the coding leptin sequence. In addition, the sequence part has a inducible T7-lac promoter (BBa_R0184 → BBa_K2406020) and a double terminator (B0010-B0012 → BBa_B0015) rrnBT1-T7TE from E. coli using RFC[10]. For its purification was used a C-terminal HIS tag. 3. A simulation in I-Tasser was used to prove the stability of the protein and also, to observe the interaction of Leptin with chitosan, was necessary the implementation of the math model created by the iGEM-TEC CEM team.