Part:BBa_K1714006

pBad/araC-RBS B0034-Ag43 thanatin 3mer as alpha domain-double terminator

Ag43 is a kind of outer membrane protein that is composed of some domains. First one is signal peptide that transport Ag43 to outer membrane. After transportation, the signal peptide is cleaved and left at inner membrane. Second one is alpha domain that has connection peptide to another Ag43 alpha domain. Last one is beta domain that has beta barrel structure to let the barrel there. Due to the function of a part of beta domain, called auto-chaperon domain, alpha domain is let outside of the membrane. This part has thanatin, a kind of antimicrobial peptide 3mer as alpha domain. This thanatin has Asp(Aspartic acid) as the last residue. So if thanatin domain is digested by AspN endoproteinase (NEB), thanatin regain own toxicity.

Experiments

Thanatin multimerization

How can we create thanatin multimer and β domain fusion protein correctly and securely? One of some methods to create thanatin multimer is inserting thanatin fragment into thanatin - Ag43 fusion protein. Once we have got thanatin monomer inserted auto-transporter biodevice, we can make thanatin multimer and Ag43 fusion protein from this plasmid. Thanatin-monomer-inserted auto-transporter biodevice contains BamHI/BglII scar (between signal peptide and thanatin) and BglII restriction enzyme site (between thanatin and β domain). So, after cutting thanatin-inserted auto-transporter with BglII, ligation this plasmid with thanatin fragment (BamHI/BglII cut) would make it possible to create thanatin multimer (Fig. 8). However, in this method, there is a possibility of reverse insertion of thanatin because C-terminal BglII of thanatin might be ligated with C-terminal BglII of auto-transporter.

Fig. 8 Insertion of thanatin into auto-transporter biodevice. (a)Forward insertion. It is desired pattern of insertion. (b)Reverse insertion. This insertion will happen by accident. We cannot insert thanatin only in forward direction selectively.

To prevent thanatin from reverse-insertion, we utilized another method. Fig. 9 shows the outline of alternative thanatin multimerization method.

Fig. 9 Flowchart of thanatin multimerization. (a)First, we amplified 2 types of fragment with 2 different primer sets. (b)Second, we digested 1st fragment (BamHI / SpeI cut) and 2nd fragment (BglII / SpeI cut). And finally, we ligated these 2 fragments. (c)Complete form of thanatin multimer secretion system.

1. Amplify the auto-transporter plasmid containing 1 thanatin by PCR with 2 primer sets below.

   • 1st primer set: for making 1st fragment

     • Forward: a primer binding to P_BAD region, which has an ability to regenerate BamHI restriction enzyme site.

     • Reverse: a primer binding to 200 bp-downstream region of suffix.

   • 2nd primer set: for making 2nd fragment

     • Forward: a primer binding to 100 bp-upstream region of suffix.

     • Reverse: a primer binding to 200 bp-downstream region of β domain.

2. Digest the amplified 2 fragments. Cut the 1st fragment with BamHI and SpeI, and cut the 2nd fragment with SpeI and BglII.

3. Ligate 1st and 2nd fragment and complete the creation of thanatin-multimer.

4. Repeat these experimental operation to increase the number of inserted thanatin.

Result

Even if we hoped that E. coli produce thanatin, it is painful task for them to do so owing to its broad-spectrum toxicity (1). Produced active thanatin would kill its host cells. In order to yield thanatin, we designed auto-transporter biodevice containing thanatin-trimer and introduced it into E. coli. After enough cultivation (10 hours) in liquid lysogeny broth (LB) medium, we resuspended cells in LB medium to optical density at 600 nm (OD₆₀₀) =0.1 (total volume of LB: 2ml). Before initiating cultivation, 200 µL of L-arabinose (final concentration; 0.1%) was added for P_BAD induction. Then, we measured temporal change of OD₆₀₀ every hour over 10 hours (until cultured cell reached stationary phase). We also determined OD₆₀₀ of E. coli containing auto-transporter biodevice alone as negative control. This control is for examination of the influence of expression of Ag43. In addition, we prepared 5 types of E. coli above without L-arabinose adding in order to examine whether gene expression is correctly induced. (Fig.10).

(c)Growth curve of transformant expressing thanatin-trimer fused with Ag43

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1612
  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1894
  Illegal NgoMIV site found at 2404
  Illegal NgoMIV site found at 2425
  Illegal AgeI site found at 979
  Illegal AgeI site found at 1413
  Illegal AgeI site found at 1485
  Illegal AgeI site found at 1557
  Illegal AgeI site found at 2164
  Illegal AgeI site found at 2578
  Illegal AgeI site found at 2820
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961