Difference between revisions of "Part:BBa K098987:Design"
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This is a mutagenized form because the original product had an extra PstI site between the repressor and terminator. While there are still extra basepairs between the end of the repressor sequence and the Biobricks scar, they no longer code for a PstI site. | This is a mutagenized form because the original product had an extra PstI site between the repressor and terminator. While there are still extra basepairs between the end of the repressor sequence and the Biobricks scar, they no longer code for a PstI site. | ||
| − | The extra base pairs are: [repressor coding region] CTCCAGCGGCCGC [Biobricks scar] | + | The extra base pairs are: [repressor coding region] CTCCAGCGGCCGC [Biobricks scar] |
| − | + | ||
| + | ===Induction Test Data=== | ||
| + | ====Experimental Design==== | ||
| + | [[Image:Thermo.png|thumb|150px|Thermo Induction Experimental Design]]<br> | ||
| + | Starter cultures of E. coli with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K098987| BBa_K098987] were grown overnight. They were then diluted and grown to OD 0.2 before separation into induced (40 ºC) and uninduced cultures (30 ºC). OD and GFP readings were taken at time 0, 2, and 4 hours. Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in their respective incubators until the end of the experiment, when OD and GFP readings were taken. | ||
| + | ====Results==== | ||
| + | Slight induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. However, since levels of GFP expression in the GFP control ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]) went down in samples at 40 ºC, it is possible that the heat affects the ability of constitutive GFP reporters to generate GFP expression. This makes it unclear how much GFP expression is actually induced by the temperature change. A slight decrease in baseline fluorescence was also observed in negative control cells ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]). <br> | ||
| + | [[Image:Thermo125.jpg|900px|Thermo Inducibility Results]]<br> | ||
| + | Results from the 1000x dilutions were inconclusive because the E. coli grows much slower at 30 ºC than at 40 ºC, so by the end of the experiment, there was a vast difference in cell concentration between the two sets of samples. | ||
===Source=== | ===Source=== | ||
Latest revision as of 06:53, 29 October 2008
temperature sensitive cI inducible system with GFP reporter and low promoter
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1600
Design Notes
This is a mutagenized form because the original product had an extra PstI site between the repressor and terminator. While there are still extra basepairs between the end of the repressor sequence and the Biobricks scar, they no longer code for a PstI site.
The extra base pairs are: [repressor coding region] CTCCAGCGGCCGC [Biobricks scar]
Induction Test Data
Experimental Design
Starter cultures of E. coli with BBa_K098987 were grown overnight. They were then diluted and grown to OD 0.2 before separation into induced (40 ºC) and uninduced cultures (30 ºC). OD and GFP readings were taken at time 0, 2, and 4 hours. Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in their respective incubators until the end of the experiment, when OD and GFP readings were taken.
Results
Slight induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. However, since levels of GFP expression in the GFP control (BBa_K098991) went down in samples at 40 ºC, it is possible that the heat affects the ability of constitutive GFP reporters to generate GFP expression. This makes it unclear how much GFP expression is actually induced by the temperature change. A slight decrease in baseline fluorescence was also observed in negative control cells (BBa_K098981).
Results from the 1000x dilutions were inconclusive because the E. coli grows much slower at 30 ºC than at 40 ºC, so by the end of the experiment, there was a vast difference in cell concentration between the two sets of samples.
Source
PGW7 plasmid and Biobricks parts.