Part:BBa_K3956014

cpTEF_6-I, scl2, PRM9

This is the full device used in CCA_San__Diego 2021's project to express scl2, a bacterial collagen. It consists of a constitutive synthetic promoter (cpTEF_6-I), the scl2 construct, and the PRM9 terminator.

Usage and Biology

Scl, streptococcal collagen-like protein, is a bacterial collagen that does not contain hydroxyproline, but is structurally similar to human collagen. Most types of scl proteins, including Scl2, are naturally expressed in the human pathogen, Streptococcus pyogenes, also known as Group A streptococcal (GAS). The Scl proteins are virulence factors of S. pyogenes and contribute to the formation of biofilm adhesion and infection of different species (Squeglia et al 14). The Scl2 gene itself is made of several parts: the V domain, the triple helical CL domain, and the tail.

Choosing the optimal promoters and terminators are crucial in initiating expression of the Scl2 construct, especially since we chose to express a bacterial protein in a eukaryotic organism. Although promoters are well-known to initiate enzyme docking and expression of a recombinant gene, the careful consideration of terminators is often neglected due to a common assumption that the sequence after the gene and the poly-A-tail folding is unimportant. However, a study by Curran et. al, 2013, challenged this assumption by demonstrating a significantly (3.5x) higher expression of fluorescent proteins when high-capacity yeast terminators were used. Because of this, we decided to choose a high-capacity natural yeast terminator correlated with the strongest expression from their paper, PRM9, which was first documented by Miller et. al, 2011.

To choose our promoter, we had several criteria we wanted to fulfill: the promoter should exhibit higher protein expression compared to commonly used promoters, and it should be constitutive and synthetic. After searching the literature on the topic, we found that the synthetic TEF1-derived promoters documented by Decoene et. al, 2019, fulfilled all of our criteria. As TEF1 is a well-characterized strong promoter in S. cerevisiae, the study authors randomized their elucidated sequence of the TEF1 minimal core promoter. They also discovered that an upstream activating sequence (UAS) around 100 bp in length dramatically improved fluorescence. The UAS is thought to be where transcription factors may bind to increase docking to the core promoter, where RNA polymerase II initiates transcription. In addition, a 5’UTR sequence usually located after the core promoter was included, as it is known to play a major role in the translation initiation process.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1192
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 598