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Team:DTU-Denmark/Methods/Visualizing GFP in the periplasm
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This is a guide on how to use biobrick [http://parts.igem.org/Part:BBa_K1067009 BBa_K1067009] to acquire pictures of GFP exported into the periplasm. The guide can also be used if having GFP fused proteins directed to the periplasm with the Twin Arginine Translocation pathway (TAT). The guide is inspired by Skoog, Karl, et al. | This is a guide on how to use biobrick [http://parts.igem.org/Part:BBa_K1067009 BBa_K1067009] to acquire pictures of GFP exported into the periplasm. The guide can also be used if having GFP fused proteins directed to the periplasm with the Twin Arginine Translocation pathway (TAT). The guide is inspired by Skoog, Karl, et al. | ||
| - | The | + | The produre is as follows: |
*TOP10 ''E. coli'' was transformed with the constructed plasmid and plated on LB agar plates. | *TOP10 ''E. coli'' was transformed with the constructed plasmid and plated on LB agar plates. | ||
*Colonies was visually inspected the day after. All colonies should be white. | *Colonies was visually inspected the day after. All colonies should be white. | ||
*The plate was induced with arabinose by using an atomizer with a 1%w/vol sterile arabinose solution. | *The plate was induced with arabinose by using an atomizer with a 1%w/vol sterile arabinose solution. | ||
*The day after colonies where visually inspected under brief exposure to UV light; a colony with a yellowish red where picked and cultured in LB containing 0.5%w/vol arabinose. The culture volume was 50mL and it was kept at 37C with shake until it reached saturation. | *The day after colonies where visually inspected under brief exposure to UV light; a colony with a yellowish red where picked and cultured in LB containing 0.5%w/vol arabinose. The culture volume was 50mL and it was kept at 37C with shake until it reached saturation. | ||
| - | *The culture was harvested by centrifugation at 5000g for 10 min at | + | *The culture was harvested by centrifugation at 5000g for 10 min at 4<sup>o</sup>C. Supernatant was discarded and the pellet was washed thoroughly by washing twice with fresh LB with no arabinose. |
*The pellet was resuspended in 25mL fresh LB with no arabinose and incubated additionally 3 hours and 40 min. to chase the cytoplasmic GFP to the periplasm. The incubation was done at 37C with shake. | *The pellet was resuspended in 25mL fresh LB with no arabinose and incubated additionally 3 hours and 40 min. to chase the cytoplasmic GFP to the periplasm. The incubation was done at 37C with shake. | ||
*Then the cells were harvested by taking 0.5mL into a centrifuge tube and adding a pinch of low melting point agarose. This was incubated at 50C for 10 min interrupted by vortexing middle ways. | *Then the cells were harvested by taking 0.5mL into a centrifuge tube and adding a pinch of low melting point agarose. This was incubated at 50C for 10 min interrupted by vortexing middle ways. | ||
| - | *The sample was vortexed and 20uL was loaded on a microscope slide and covered with a cover slip. This could then be stored at | + | *The sample was vortexed and 20uL was loaded on a microscope slide and covered with a cover slip. This could then be stored at 4<sup>o</sup>C or immediately examined. |
*Pictures were acquired on fluorescents microscope with 100x lens. Both GFP and RFP filters was used. To get the clear difference between periplasm and cytoplasm 2 pictures with each filter was taken; one with short exposure time and one with long. The short exposure time was subtracted from the long and finally the GFP and RFP pictures were merged to get RFP as a cytoplasmic contrast to the periplasmic GFP. | *Pictures were acquired on fluorescents microscope with 100x lens. Both GFP and RFP filters was used. To get the clear difference between periplasm and cytoplasm 2 pictures with each filter was taken; one with short exposure time and one with long. The short exposure time was subtracted from the long and finally the GFP and RFP pictures were merged to get RFP as a cytoplasmic contrast to the periplasmic GFP. | ||
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==References== | ==References== | ||
Latest revision as of 18:07, 4 October 2013
Periplasmic GFP
Guide on how to visualize GFP in the periplasm
This is a guide on how to use biobrick [http://parts.igem.org/Part:BBa_K1067009 BBa_K1067009] to acquire pictures of GFP exported into the periplasm. The guide can also be used if having GFP fused proteins directed to the periplasm with the Twin Arginine Translocation pathway (TAT). The guide is inspired by Skoog, Karl, et al.
The produre is as follows:
- TOP10 E. coli was transformed with the constructed plasmid and plated on LB agar plates.
- Colonies was visually inspected the day after. All colonies should be white.
- The plate was induced with arabinose by using an atomizer with a 1%w/vol sterile arabinose solution.
- The day after colonies where visually inspected under brief exposure to UV light; a colony with a yellowish red where picked and cultured in LB containing 0.5%w/vol arabinose. The culture volume was 50mL and it was kept at 37C with shake until it reached saturation.
- The culture was harvested by centrifugation at 5000g for 10 min at 4oC. Supernatant was discarded and the pellet was washed thoroughly by washing twice with fresh LB with no arabinose.
- The pellet was resuspended in 25mL fresh LB with no arabinose and incubated additionally 3 hours and 40 min. to chase the cytoplasmic GFP to the periplasm. The incubation was done at 37C with shake.
- Then the cells were harvested by taking 0.5mL into a centrifuge tube and adding a pinch of low melting point agarose. This was incubated at 50C for 10 min interrupted by vortexing middle ways.
- The sample was vortexed and 20uL was loaded on a microscope slide and covered with a cover slip. This could then be stored at 4oC or immediately examined.
- Pictures were acquired on fluorescents microscope with 100x lens. Both GFP and RFP filters was used. To get the clear difference between periplasm and cytoplasm 2 pictures with each filter was taken; one with short exposure time and one with long. The short exposure time was subtracted from the long and finally the GFP and RFP pictures were merged to get RFP as a cytoplasmic contrast to the periplasmic GFP.
References
- Skoog, Karl, et al. "Sequential Closure of the Cytoplasm and Then the Periplasm during Cell Division in Escherichia coli." Journal of bacteriology 194.3 (2012): 584-586.
