Part:BBa_K1962008
Colicin E9::Ssp2 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 Ssp2 from Serratia marcescens. Ssp2 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 serratia marcescens were cloned onto the C-terminal of the receptor binding domain of truncated colicins Ia and E9, by making use of the engineered multiple cloning site. Resulting in the generation of synthetic colicins col Ia-ssp2 BBa_K1962003col E9-ssp1 BBa_K1962007 and col E9-ssp2 BBa_K1962008. These were amplified with a C-terminal HA-tag and then cloned into pBAD18 vector with pBAD promoter of the araBAD operon. pBAD can be repressed with glucose and induced with arabinose. During the cloning steps 0.5% glucose was added to the cells in order to repress the expression of the synthetic colicins.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1864
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1357
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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