Difference between revisions of "Team:TecCEM/Parts"

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                         migration, proliferation and differentiation. We approached the scaffold design with three
 
                         migration, proliferation and differentiation. We approached the scaffold design with three
 
                         different proteins, each with properties to achieve better tissue regeneration and could
 
                         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.
+
                         interact with each other. This meant we needed a way to produce protein overexpression in <i>E.
−
                         coli vectors. </p>
+
                         coli</i> vectors. </p>
 
                     <p> In this section the description of the constructs and background behind the constructs is
 
                     <p> In this section the description of the constructs and background behind the constructs is
 
                         explained. </p>
 
                         explained. </p>
Line 62: Line 62:
 
                     <ol>
 
                     <ol>
 
                         <li>A collagen-like synthetic gene comprising heparin binding site of collagen type V (HepV)
 
                         <li>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
+
                             was employed. The synthetic gene for COL5A1 was optimized for codon usage in <i>E. coli</i>
 
                             Rosetta 2 (DE3), whereas illegal sequences were removed.</li>
 
                             Rosetta 2 (DE3), whereas illegal sequences were removed.</li>
−
                         <li>The nucleotide sequence of collagen is optimized for E. coli Rosetta 2 (DE3) and cloned
+
                         <li>The nucleotide sequence of collagen is optimized for <i>E. coli</i> Rosetta 2 (DE3) and cloned
 
                             into the backbone pSB1C3 that consists of a chloramphenicol resistant gene using EcoRI and
 
                             into the backbone pSB1C3 that consists of a chloramphenicol resistant gene using EcoRI and
 
                             PstI restriction sites, with an inducible T7-lac promoter for the construction of the
 
                             PstI restriction sites, with an inducible T7-lac promoter for the construction of the
Line 89: Line 89:
 
                             and tenascin is involved in affinity with many growth factors, specifically with Fibroblast
 
                             and tenascin is involved in affinity with many growth factors, specifically with Fibroblast
 
                             Growth Factor (FGF). </li>
 
                             Growth Factor (FGF). </li>
−
                         <li>The nucleotide sequence of TNCIII5 is optimized for E. coli BL21 DE3 and cloned into the
+
                         <li>The nucleotide sequence of TNCIII5 is optimized for <i>E. coli</i> BL21 DE3 and cloned into the
 
                             backbone (pSB1C3) using EcoRI and PstI restriction sites, with an inducible T7-lac promoter
 
                             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
 
                             and rrnB-T1 terminator using RFC[10]. The domain was expressed by bacterial expression as
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                             composed by Tenascin, collagen, and heparin. Leptin protein is the product of the ob gene
 
                             composed by Tenascin, collagen, and heparin. Leptin protein is the product of the ob gene
 
                             and it is found in adipocytes cells. </li>
 
                             and it is found in adipocytes cells. </li>
−
                         <li>The coding sequence is optimized for Escherichia coli BL21 (DE3) and cloned in a pSB1C3
+
                         <li>The coding sequence is optimized for <i>Escherichia coli</i> BL21 (DE3) and cloned in a pSB1C3
 
                             plasmid. To get the cloned sequence is necessary the implementation of EcoRI and PstI
 
                             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
 
                             restriction sites, that they are located in the prefix and suffix, respectively, previously
 
                             sintetized for the coding leptin sequence. In addition, the sequence part has an inducible
 
                             sintetized for the coding leptin sequence. In addition, the sequence part has an inducible
 
                             T7-lac promoter (BBa_R0184 → BBa_K2406020) and a double terminator (B0010-B0012 →
 
                             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
+
                             BBa_B0015) rrnBT1-T7TE from <i>E. coli</i> using RFC[10]. For its purification was used a
 
                             C-terminal HIS tag. </li>
 
                             C-terminal HIS tag. </li>
 
                         <li>A simulation in I-Tasser was used to prove the stability of the protein and also, to
 
                         <li>A simulation in I-Tasser was used to prove the stability of the protein and also, to
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                                 <td><a href="http://parts.igem.org/Part:BBa_K2719003">BBa_K2719003</a></td>
 
                                 <td><a href="http://parts.igem.org/Part:BBa_K2719003">BBa_K2719003</a></td>
 
                                 <td>Tag</td>
 
                                 <td>Tag</td>
−
                                 <td>GST (Optimized for E.coli BL21)</td>
+
                                 <td>GST (Optimized for <i>E.coli</i> BL21)</td>
 
                                 <td>Karla Soto Blas</td>
 
                                 <td>Karla Soto Blas</td>
 
                             </tr>
 
                             </tr>

Revision as of 00:40, 18 October 2018

Cell Gif

Parts Overview

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 of 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 an 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.
IMP-1
Figure 1. Collagen construct pSB1C3

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 bound 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 sintetized for the coding leptin sequence. In addition, the sequence part has an 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.
IMP-1
Figure 3. Leptin construct pSB1C3
Name Type Description Designers
BBa_K2719000 Tag GST Karla Soto Blas
BBa_K2719001 Coding Tenascin Domain V Karla Soto Blas
BBa_K2719002 Coding GST + Tenascin Domain V Karla Soto Blas
BBa_K2719003 Tag GST (Optimized for E.coli BL21) Karla Soto Blas
BBa_K2719004 Coding Tenascin Fibronectin Domains (I-V) Karla Soto Blas
BBa_K2719005 Device Tenascin Domain V Expression Device Karla Soto Blas
BBa_K2719006 Coding Collagen V - Like Armando Cortés Reséndiz & María José Ugarte Orozco
BBa_K2719007 Device Collagen V - Like Expression Device. Armando Cortés Reséndiz & María José Ugarte Orozco
BBa_K2719008 Coding Leptin Andrea Pamela Jimenez Tapia & Rodrigo Valencia Ocampo
BBa_K2719009 Device Leptin Coding Device Andrea Pamela Jimenez Tapia & Rodrigo Valencia Ocampo
BBa_K2719010 Coding Leptin codiNg device with blue chromoprotein Juan Carlos Rueda Silva & Arantxa Karam Coppola
BBa_K2719011 Coding Collagen coding device with blue chromoprotein Juan Carlos Rueda Silva & Arantxa Karam Coppola
BBa_K2719012 Coding TCD5 coding device with blue chromoprotein Juan Carlos Rueda Silva & Arantxa Karam Coppola
BBa_K2719013 Device Blue Chromoprotein and RFP combined reporter Juan Carlos Rueda Silva & Arantxa Karam Coppola