Part:BBa_K531007

P_rsaA constitutive promoter, esp optimized for Caulobacter, and rsaA

P_rsaA constitutive promoter from Caulobacter crescentus, esp, a suspected serine protease from Staphylococcus epidermidis, optimized for expression in Caulobacter crescentus, and the C-terminal secretion tag from rsaA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 321
  Illegal XhoI site found at 891
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1146
  Illegal AgeI site found at 1269
  
• 1000
  COMPATIBLE WITH RFC[1000]

The following results show that most of our chimeric RsaA-fusion proteins were expressed and secreted from Caulobacter, indicating that the rsaA secretion tag works.

Figure 1. Expression and secretion of chimeric proteins from Caulobacter. We cultured each of our Caulobacter secretion strains and collected the supernatants from equal numbers of cells. The proteins in the supernatants were precipitated with TCA, run on a 6-18% polyacrylamide gel, and silver stained. Results show that proteins of the expected size were secreted from PrsaA with chimeric Esp/RsaA C-terminal construct. However, even though we used gradient gel and silver staining, bands did not appear easily: only those constructs that presumably had a high yield of heterologous protein had bands corresponding to the desired biofilm inhibitor protein.

Figure 2. Expression and secretion of chimeric proteins from Caulobacter. We cultured each of our Caulobacter secretion strains and collected the supernatants from equal numbers of cells. The proteins in the supernatants were precipitated with TCA, run on a 6-18% polyacrylamide gel, and silver stained. Results show that proteins of the expected size were secreted from the following strains: both DspB and Esp constructs with Pxyl inducible promoter and chimeric Esp with promoter BBa_K081005.

Figure 3. Relative S. aureus biofilm growth after treatment with different engineered Caulobacter strains (three letters abbreviation for constructs: B for BBa_K081005, R for PrsaA, X for Pxyl, O for optimized, W for wild type, E for Esp, D for DspB, and CC for WT Caulobacter crescentus). Relative biofilm growth is calculated by subtracting the ODmean of S. aureus only treatment from the OD of each well.

Figure 4. Here the standard group was composed of S. aureus only and WT groups. Both DspB and Esp can inhibit the biofilm growth. Though not statistically significant, the biofilm assay data showed a general trend that Esp, which is known to target S. aureus biofilm specifically, has a better inhibition effect than DspB, which is generally targeting most biofilms.

Figure 5. All promoters were working great in Caulobacter. No significant difference among three promoters were shown.