Part:BBa_K2029015

pSBbs1AK1 (replicative Bacillus subtilis expression vector)

This part is a dual expression vector for Bacillus subtilis and Escherichia coli. It consists of an origin of replication for both E. coli and B. subtilis, allowing for propagation in both organisms. Selection is possible via antibiotic resistance for kanamycin in B. subtilis and for ampicillin in E. coli.

Miniprep in E.coli

pSBbs1AK1 was succesfully transformed into E. coli. The backbone confered both ampicillin and kanamycin reistance to E. coli as indicated by its growth in LB medium containing 100mg/mL ampicillin and 50mg/mL kanamycin. A restriction analysis of DNA isolated from the culture comfirmed the presence of our backbone.

Restriction analysis of pSBbs1AK1 showing linearisation of the plasmid when cut with the BioBrick sites E, P, X, S, as well as with KpnI and HindIII

B.subtilis

pSBbs1AK1 was also transformed into B. subtilis. Growth of colonies on 50mg/mL kanamycin agarose plates (Table 1) as well as in liquid cultures containing 50mg/mL kanamycin indicate that pSBbs1AK1 transfers kanamycin antibiotic resistance within B. subtilis. Since the isolation of DNA from B. subtilis was difficult due to the high nuclease activity, the presence of the backbone in the kanamycin resistant B. subtilis colonies could not be confirmed via restriction analysis.

600ng of pSBbs1AK1 and 60ng of pDG148_engB were transformed into 1 mL of competent cells via electroporation. The agarose plates were cultured overnight at 37°C

DNA-concentration (ng/μl)	Number of colonies
10	11
5	3
1	1
0	0

Table 1

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5414
  Illegal XbaI site found at 5429
  Illegal SpeI site found at 1
  Illegal PstI site found at 15
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5414
  Illegal SpeI site found at 1
  Illegal PstI site found at 15
  Illegal NotI site found at 8
  Illegal NotI site found at 5420
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5414
  Illegal BglII site found at 4205
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 5414
  Plasmid lacks a suffix.
  Illegal XbaI site found at 5429
  Illegal SpeI site found at 1
  Illegal PstI site found at 15
  Illegal AgeI site found at 3816
  Illegal AgeI site found at 4778
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 1176
  Illegal BsaI.rc site found at 2531
  Illegal BsaI.rc site found at 5047
  Illegal SapI.rc site found at 4029