Difference between revisions of "Part:BBa K847002:Experience"
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'''Assay''' <BR>
 
'''Assay''' <BR>
 
''Radiation''<BR>
 
''Radiation''<BR>
−
Liquid cultures of NEB5α E. coli transformed with  DpsG and negative control were grown up over night at 37°C. The following day, the cells were washed and resuspended in 0.9% NaCl solution. Cell concentration was then adjusted to 10^7/mL in 5mL of a glass Petri dish. Each sample was then exposed to 1.2 J/(m^2*sec) of UV-C radiation from a UV lamp for a cumulative of 0 seconds, 2 seconds, 5 seconds, 10 seconds, 20 seconds, and 30 seconds. After each exposure, a dilution spot assay was conducted to determine the final number of surviving cells.  <BR>
+
Liquid cultures of NEB5α E. coli transformed with  DpsMP1 and negative control were grown up over night at 37°C. The following day, the cells were washed and resuspended in 0.9% NaCl solution. Cell concentration was then adjusted to 10^7/mL in 5mL of a glass Petri dish. Each sample was then exposed to 1.2 J/(m^2*sec) of UV-C radiation from a UV lamp for a cumulative of 0 seconds, 2 seconds, 5 seconds, 10 seconds, 20 seconds, and 30 seconds. After each exposure, a dilution spot assay was conducted to determine the final number of surviving cells.  <BR>
 
''Base''<BR>
 
''Base''<BR>
 
Liquid cultures of negative control and DpsG transformed NEB5α E. coli were grown up over night at 37°C. After incubation, LB + amp media of pH 3.54, 4.60, 6.93, 9.07, 9.50 were created using HCl and NaOH. In a 96 well plate, 8 wells were filled with LB + amp blank. Next, 5uL of each transformed bacteria was placed in five different pH wells. This was replicated four times, thus, creating a 96 well plate with 8 blanks, and four replicates of each test construct at five different pH’s. Finally, the OD600 was measured every five minutes for 24 hours while the plate was incubated at 37°C, shook for 1 minute before every sample and between every sample in a spectrophotometer. <BR><BR>
 
Liquid cultures of negative control and DpsG transformed NEB5α E. coli were grown up over night at 37°C. After incubation, LB + amp media of pH 3.54, 4.60, 6.93, 9.07, 9.50 were created using HCl and NaOH. In a 96 well plate, 8 wells were filled with LB + amp blank. Next, 5uL of each transformed bacteria was placed in five different pH wells. This was replicated four times, thus, creating a 96 well plate with 8 blanks, and four replicates of each test construct at five different pH’s. Finally, the OD600 was measured every five minutes for 24 hours while the plate was incubated at 37°C, shook for 1 minute before every sample and between every sample in a spectrophotometer. <BR><BR>

Revision as of 02:42, 4 October 2012


Assay
Radiation
Liquid cultures of NEB5α E. coli transformed with DpsMP1 and negative control were grown up over night at 37°C. The following day, the cells were washed and resuspended in 0.9% NaCl solution. Cell concentration was then adjusted to 10^7/mL in 5mL of a glass Petri dish. Each sample was then exposed to 1.2 J/(m^2*sec) of UV-C radiation from a UV lamp for a cumulative of 0 seconds, 2 seconds, 5 seconds, 10 seconds, 20 seconds, and 30 seconds. After each exposure, a dilution spot assay was conducted to determine the final number of surviving cells.
Base
Liquid cultures of negative control and DpsG transformed NEB5α E. coli were grown up over night at 37°C. After incubation, LB + amp media of pH 3.54, 4.60, 6.93, 9.07, 9.50 were created using HCl and NaOH. In a 96 well plate, 8 wells were filled with LB + amp blank. Next, 5uL of each transformed bacteria was placed in five different pH wells. This was replicated four times, thus, creating a 96 well plate with 8 blanks, and four replicates of each test construct at five different pH’s. Finally, the OD600 was measured every five minutes for 24 hours while the plate was incubated at 37°C, shook for 1 minute before every sample and between every sample in a spectrophotometer.


Results
Radiation: Characterized; however, further testing needed. Refer to: http://2012.igem.org/Team:Stanford-Brown/HellCell/Radiation. Base: Works. Refer to: http://2012.igem.org/Team:Stanford-Brown/HellCell/pH


Applications of BBa_K847002

Astrobiology revolves around three central questions: "Where do we come from?", "Where are we going?", and "Are we alone?" To approach the second question, the Hell Cell subgroup of the Stanford-Brown iGEM team developed BioBricks that allow a cell to survive harsh extraterrestrial conditions. Such a toolset could create a space-ready synthetic organism to perform useful functions off-world. This gene is one of the toolset, potentially conferring radiation resistance to otherwise radiation-intolerant bacteria.

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