Difference between revisions of "Team:UNSW Australia/Basic Part"

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<li>Gibson, D. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nature Methods</i> 6, 343-345 (2009).</li>
 
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<li>Vellanoweth, R. & Rabinowitz, J. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and <i>Escherichia coli</i> in vivo. <i>Molecular Microbiology</i> 6, 1105-1114 (1992).</li>
 
<li>Vellanoweth, R. & Rabinowitz, J. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and <i>Escherichia coli</i> in vivo. <i>Molecular Microbiology</i> 6, 1105-1114 (1992).</li>
 
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Revision as of 23:59, 17 October 2018

Basic Part

Abstract

The UNSW iGEM team successfully created RFC10 compatible BioBricks linked to our project. BioBricks were validated by sequence verification and a diagnostic gel, characterised experimentally, and physically submitted to the registry. The BioBricks we are submitting for silver are BBa_K2710000 and BBa_K2710001, which encode for the Alpha and Beta subunits of the Prefoldin protein respectively, and BBa_K2710002, His-Alpha Prefoldin with a SpyCatcher. Refer to the parts registry pages for more details.

Best Basic Part

This year, the UNSW iGEM team has chosen to submit the His-Alpha Prefoldin with SpyCatcher part (BBa_K2710002) for the best basic part award.

Figure 1: Diagram illustrating the process of Gibson assembly sequence insertion into the plasmid vector1.

Our DNA constructs were cloned into pETDuet-1 and pRSFDuet-1 plasmid vectors, as well as pET-19b in our later experiments. The Duet vectors carry two expression units that are controlled by a T7-lac promoter and terminator for protein expression. The Duet plasmids, pETDuet-1 and pPRSFDuet-1, both possess an ampicillin and kanamycin resistance gene, respectively. Meanwhile pET-19 confers ampicillin resistance (Figure 2). These specific vectors were chosen so that the prefoldin-catcher and enzyme-tag DNA constructs could be cloned into the same cell, allowing the entire scaffold to be expressed simultaneously. Furthermore, pETDuet-1 and pRSFDuet-1 plasmids possess different origins of replication, which enables in vivo production of the scaffold-enzyme complex.

Figure 2: Plasmid maps depicting pETDuet-1, pRSFDuet-1 and pET19-b. Resistance genes are shown in red. Images were generated by Benchling.

References

  1. Gibson, D. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343-345 (2009).
  2. Siegert, R., Leroux, M., Scheufler, C., Hartl, F. & Moarefi, I. Structure of the Molecular Chaperone Prefoldin. Cell 103, 621-632 (2000).
  3. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109, E690-E697 (2012).
  4. Vellanoweth, R. & Rabinowitz, J. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Molecular Microbiology 6, 1105-1114 (1992).