Part:BBa_K1962007

Colicin E9::Ssp1 Chimera

This part contains an translational fusion protein between the truncated DNase-domain-lacking version of Colicin E9 (BBa_K1962006) and an antibacterial toxin (peptidoglycan hydrolase) called Ssp1 from Serratia marcescens. Ssp1 is an antibacterial effector normally secreted by the Type VI secretion system (T6SS) by Serratia marcescens and it has peptidoglycan endopeptidase activity.

Usage and Biology

Ssp1 and Ssp2 from S .marcescens were cloned onto multiple cloning sites on the C-terminal of the receptor binding domain of truncated colicins Ia and E9, resulting in modified col Ia-ssp2, col E9-ssp1 and col E9-ssp2. These were fused with a HA-tag then cloned into pBAD18 vector with pBAD promoter of the araBAD operon. pBAD can be repressed with glucose and induced with arabinose. The modified colicins were ligated with pBAD18 and grown on LB plates with kanamycin resistance and 0.5% glucose, to repress expression of the toxin while it is taken up by cells.

Western blots were carried out to determine if our fusion proteins were being expressed (Fig 1).

Figure 1: Detection of Ha-tagged pcol E9-ssp1 and ssp2: Single colonies of pcol E9-ssp1 and pcol E9-ssp2 were grown overnight in 5ml LB with 0.5% glucose at 37°C. 250μl of cells were then grown in 25ml of LB at 37°C until they reached an OD of 0.5A. They were then represssed with 0.2% glucose and induced with 0.2% arabinose and 0.5% arabinose and incubated at 37°C for 5 hours. 2ml sample of each was taken, pelleted, then 100μl of laemmli sample buffer and B-mercaptoethanol was added to each and they were boiled for 10 mins. 20μl of samples were separated by SDS-PAGE (10% acrylamide) transferred to membrane and probed with HA-antibody. pcol E9-ssp1 and pcol E9-ssp2 were expressed when induced with arabinose at both concentrations.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1879
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1357
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1543
  
• 1000
  COMPATIBLE WITH RFC[1000]