Difference between revisions of "Part:BBa K1962003"
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Usage and Biology | Usage and Biology | ||
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| + | 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). | ||
https://static.igem.org/mediawiki/parts/8/8a/Coliassp2frulhuq.png | https://static.igem.org/mediawiki/parts/8/8a/Coliassp2frulhuq.png | ||
| + | Figure 1: Detection of Ha-tagged pcol Ia-ssp2: Single colonies of pcol Ia-MCS and pcol Ia-ssp2 2:1 and 3:1 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. 1ml 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. 5μl of pre-induced and induced samples were separated by SDS-PAGE (10% acrylamide) transferred to membrane and probed with HA-antibody. Expression of pcol Ia-ssp2 was detected when induced by both concentrations of arabinose. | ||
Revision as of 21:14, 22 October 2016
Colicin Ia::Ssp2 Chimera
This part contains an translational fusion protein between the truncated bacteriocin-free version of Colicin Ia (BBa_K1962002) 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) in Serratia marcescens and it has peptidoglycan endopeptidase activity, degrading peptidoglycan in the periplasm of the target cell where it cleaves between y-D-glutamic acid and L-mesodiaminopimelic acid in peptidoglycan.
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 Ia-ssp2: Single colonies of pcol Ia-MCS and pcol Ia-ssp2 2:1 and 3:1 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. 1ml 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. 5μl of pre-induced and induced samples were separated by SDS-PAGE (10% acrylamide) transferred to membrane and probed with HA-antibody. Expression of pcol Ia-ssp2 was detected when induced by both concentrations of arabinose.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1858
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1351
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]