Cconboy:Promoter Characterization/Discussion: Difference between revisions
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===Characterization of Promoters=== | ===Characterization of Promoters=== | ||
* In characterizing constitutive transcriptional promoters, there are at least | * In characterizing the performance of constitutive transcriptional promoters, there are at least three parameters of interest: (1) the rate of PoPS initiated off the promoter, (2) the promoter's metabolic load or burden, and (3) its genetic stability or rate of loss due to mutation. | ||
**R0052 | ** In this study we've undertaken the ''relative'' measurement of the first parameter. ((etc.)) | ||
* | ** Our results indicate that R0052 is a very strong promoter relative to the others in the Registry. Expression of YFP from R0052 gave a wide distribution of YFP levels and multiple sub populations, though sequencing the construct after the experiment indicated that it had not mutated. The increased variability in gene expression and the stressed cell morophology observed by microscopy are likely a consequence of the high metabolic load of the construct. Such a part may be strongly selected against. Although we have not measured its rate of loss directly, there is annecdotal evidence that this part is unstable. [http://parts.mit.edu/d/parts/partsdb/view.cgi?part_id=190] | ||
* We have also considered the effect of media conditions and growth phase on our part characterization. ((add discussion of two different media conditions (LB and supplemented M9) and growth phase effects.)) | |||
===Caveats=== | ===Caveats=== | ||
* GFP fluorescence is an imperfect surrogate measurement for transcription. In this study we assume they are relatively correlated, however, differences in translation rates and mRNA stability between constructs are expected to limit that correlation and the accuracy of this measurement. | * GFP fluorescence is an imperfect surrogate measurement for transcription. In this study we assume they are relatively correlated, however, differences in translation rates and mRNA stability between constructs are expected to limit that correlation and the accuracy of this measurement. | ||
** In particular, we hypothesize that transcripts of ((construct with R0053)) may have a reduced expression level compared to other constructs in this study due to local secondary structure, predicted from the free energy of folding, which may occlude the | ** In particular, we hypothesize that transcripts of ((construct with R0053)) may have a reduced expression level compared to other constructs in this study due to local secondary structure, predicted from the free energy of folding, which may occlude the transcript's ribosome binding site. ((reference, link to figure, compare to results with different RBS.)) | ||
* ((Time-course data indicates that samples were not at steady state during log-phase growth.)) | * ((Time-course data indicates that samples were not at steady state during log-phase growth.)) | ||
* MC4100 is not a good chassis for operating BBa_I0500 (pBad promoter). The feed-forward regulation of the endogenous promoter controlling expression of the arabinose transporter prevents linear induction with increasing arabinose concentration. ((Engineered strain from Keasling's lab, used by jrk for operation of the screening plasmid.)) | |||
* | ===Future Work=== | ||
* We'd like to be able to characterize the rate of transcription initiated from a promoter in terms of absolute numbers. | |||
** Check out the next phase of this research [[Cconboy: Soup to Nuts Project Description|here]]. | |||
* As synthetic biologists we'd like to have a set of promoters that are regulable and inducible as well. Characterizing these components will require additional considerations: | |||
** For inducible promoters we'd like to measure (1) the transfer function PoPS vs. inducer concentration and (2) the latency in induction. | |||
***[[PoPS device screening plasmid| Characterization of the pBad promoter transfer function.]] | |||
** For regulated promoters we'd like to measure the transfer function between rate of expression of the repressor molecule and PoPS out of the promoter. | |||
*** See Reshma Shetty's work on characterizing QPI's | |||
Latest revision as of 17:10, 22 March 2006
Page in progress
Characterization of Promoters
- In characterizing the performance of constitutive transcriptional promoters, there are at least three parameters of interest: (1) the rate of PoPS initiated off the promoter, (2) the promoter's metabolic load or burden, and (3) its genetic stability or rate of loss due to mutation.
- In this study we've undertaken the relative measurement of the first parameter. ((etc.))
- Our results indicate that R0052 is a very strong promoter relative to the others in the Registry. Expression of YFP from R0052 gave a wide distribution of YFP levels and multiple sub populations, though sequencing the construct after the experiment indicated that it had not mutated. The increased variability in gene expression and the stressed cell morophology observed by microscopy are likely a consequence of the high metabolic load of the construct. Such a part may be strongly selected against. Although we have not measured its rate of loss directly, there is annecdotal evidence that this part is unstable. [1]
- We have also considered the effect of media conditions and growth phase on our part characterization. ((add discussion of two different media conditions (LB and supplemented M9) and growth phase effects.))
Caveats
- GFP fluorescence is an imperfect surrogate measurement for transcription. In this study we assume they are relatively correlated, however, differences in translation rates and mRNA stability between constructs are expected to limit that correlation and the accuracy of this measurement.
- In particular, we hypothesize that transcripts of ((construct with R0053)) may have a reduced expression level compared to other constructs in this study due to local secondary structure, predicted from the free energy of folding, which may occlude the transcript's ribosome binding site. ((reference, link to figure, compare to results with different RBS.))
- ((Time-course data indicates that samples were not at steady state during log-phase growth.))
- MC4100 is not a good chassis for operating BBa_I0500 (pBad promoter). The feed-forward regulation of the endogenous promoter controlling expression of the arabinose transporter prevents linear induction with increasing arabinose concentration. ((Engineered strain from Keasling's lab, used by jrk for operation of the screening plasmid.))
Future Work
- We'd like to be able to characterize the rate of transcription initiated from a promoter in terms of absolute numbers.
- Check out the next phase of this research here.
- As synthetic biologists we'd like to have a set of promoters that are regulable and inducible as well. Characterizing these components will require additional considerations:
- For inducible promoters we'd like to measure (1) the transfer function PoPS vs. inducer concentration and (2) the latency in induction.
- For regulated promoters we'd like to measure the transfer function between rate of expression of the repressor molecule and PoPS out of the promoter.
- See Reshma Shetty's work on characterizing QPI's
