Part:BBa_K2924054

Hpall + SPYurl + alpha s2 + fd terminator

P_HpaII with RBS expressing SPYurl+ α-s2-casein + 6xHis-tag, terminated by the fd Terminator in Bacillus subtilis.

Usage and Biology

Fig.1: Scheme of expression construct for B. subtilis. The insert, containing the promoter P_Hpall (BBa_K2924043), α-s2-casein gene (BBa_K2924027) and the double fd terminator (BBa_K2924044), was cloned into the pBSMUl1 backbone with different secretion signals - here: SPYurl (K2924050 )

This composite part (Fig.1) contains the constitutive promoter P_Hpall (BBa_K2924043), expressing α-s2-casein (BBa_K2924027) with the secretion signal SPYurl (BBa_K2924050) and the fd terminator (BBa_K2924044) for expression and secretion of the protein in Bacillus subtilis.

B. subtilis is a frequently used expression system for secreting proteins, which can avoid some common problems with intracellular over expressions like low expression rates, improper protein-folding, formation of inclusion bodies or product toxicity. The secretion is achieved by secretion signals, which are fused to proteins, leading to an export of those tagged proteins by different mechanism, while the secretion tag is cleaved of in many cases after successful secretion.

The organism has been widely described and examined; its genome has been fully sequenced and all important genes and metabolic pathways are known. The fundamental architecture of B. subtilis cell wall can ease protein secretion pathways and allow the organism to secrete high levels of extracellular proteins directly into the medium¹. For food production, the gram positive bacterium is highly favored, given the fact that it is examined as a GRAS organism (generally recognized as safe)^2,3,4. Furthermore, compared to other organisms, it does not produce endotoxins that are wished to be removed of the final product⁵.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 1127
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 557
  

Characterization

For expression and secretion of α-s2-casein the gene was cloned into the high-copy pBSMUl1-SPNprE plasmid, N-terminally fused to the signal peptide SPYurl and C-terminally fused to a 6xHis-tag for easier purification and immunodetection.

Fig. 2: 250 ml Erlenmeyer flasks with 30 ml LB with [50 µg/mL] Kanamycin, the culture medium for the transformed B. subtilis strains.

A single positive transformant of the pBSMUl1-SPYurl+α-s2-casein plasmid was inoculated into 5 ml LB medium containing 50 µg/ mL Kanamycin and incubated at 37°C, 250 rpm for the next 24 hours. After 24 hours, 29 ml LB Medium were inoculated with 1 ml overnight culture and incubated at 37°C, 250 rpm for 48 hours. We used erlenmeyer flasks with a volume of 250 ml to ensure sufficient aeration (Fig. 2).