Difference between revisions of "Part:BBa J100534:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | We decided not to order the AHL chemical because there is no lux cassette in the E. coli cultures that we will use (not possible for the lux gene to be expressed). This lux R gene is typically expressed in other bioluminescent bacteria, including Vibrio fischeri. Therefore, we are allowing it to function as a constitutive promoter. We have two experimental groups that will be compared to a negative control, which contains water instead of the diluted oligo solution, and a positive control, which contains neither water nor the oligo solution. Spectrophotometry will be used to compare the optical density of each E. coli population and to create a ratio of red to optical density for the purpose of measuring the amount of red in each cell. This will tell us how effective the promoter is. Because we cannot work with the given repressor, we decided to alter temperature as a variable. We hypothesized that allowing the bacteria to incubate for one hour at a temperature of 42 degrees Celsius will act as an inducer for the specific promoter. We incubated samples for each of our experimental groups, which we separated into X1, X2, and X3, and then ran them in the spectrophotometer to compare the amount of fluorescence produced with the optic density of both the incubated and non-incubated groups. | + | We decided not to order the AHL chemical because there is no lux cassette in the E. coli cultures that we will use (not possible for the lux gene to be expressed). This lux R gene is typically expressed in other bioluminescent bacteria, including Vibrio fischeri. Therefore, we are allowing it to function as a constitutive promoter. We have two experimental groups that will be compared to a negative control, which contains water instead of the diluted 50 nM oligo solution, and a positive control, which contains neither water nor the oligo solution. GGA was used to insert the promoters into the experimental groups (GGA master solution, ligase, oligo, and BSA I type II restriction enzyme were added and held at different temperatures). Spectrophotometry will be used to compare the optical density of each E. coli population and to create a ratio of red to optical density for the purpose of measuring the amount of red in each cell. This will tell us how effective the promoter is. Because we cannot work with the given repressor, we decided to alter temperature as a variable. We hypothesized that allowing the bacteria to incubate for one hour at a temperature of 42 degrees Celsius will act as an inducer for the specific promoter. We incubated samples for each of our experimental groups, which we separated into X1, X2, and X3, and then ran them in the spectrophotometer to compare the amount of fluorescence produced with the optic density of both the incubated and non-incubated groups. |
| + | PCR was used to determine if the promoters were successfully cloned. PCR was done by adding PCR master mix (primer, DNA polymerase, dNTPs, and a buffer) to N and X1, X2, X3 groups. The bacteria provided the template for PCR. PCR results were run through gel electrophoresis to compare sizes. The added primer will be shorter (no longer than 60 nucleotides plus sticky ends) than the original primer in the N group that produced GFP. | ||
In moving forwards, we are currently looking into whether or not there is a gene that is similar to the lux R gene that is naturally expressed in E. coli that our promoter might be able to affect/function with. The bacteria in which this promoter is usually found also undergoes growth sue to density-based quorum sensing. To see if E. coli altered with this promoter can divide in the same way, we will take a dense cell colony, dilute it, and centrifuge it down. We will take an aliquot from the top of the centrifuged sample, which should theoretically contain some chemical that induces quorum sensing division. We will then spread that on another cell colony of the same dilution and see if it promotes growth. | In moving forwards, we are currently looking into whether or not there is a gene that is similar to the lux R gene that is naturally expressed in E. coli that our promoter might be able to affect/function with. The bacteria in which this promoter is usually found also undergoes growth sue to density-based quorum sensing. To see if E. coli altered with this promoter can divide in the same way, we will take a dense cell colony, dilute it, and centrifuge it down. We will take an aliquot from the top of the centrifuged sample, which should theoretically contain some chemical that induces quorum sensing division. We will then spread that on another cell colony of the same dilution and see if it promotes growth. | ||
Revision as of 16:37, 10 March 2020
Constitutive RFP Promoter in E. coli
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 35
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We decided not to order the AHL chemical because there is no lux cassette in the E. coli cultures that we will use (not possible for the lux gene to be expressed). This lux R gene is typically expressed in other bioluminescent bacteria, including Vibrio fischeri. Therefore, we are allowing it to function as a constitutive promoter. We have two experimental groups that will be compared to a negative control, which contains water instead of the diluted 50 nM oligo solution, and a positive control, which contains neither water nor the oligo solution. GGA was used to insert the promoters into the experimental groups (GGA master solution, ligase, oligo, and BSA I type II restriction enzyme were added and held at different temperatures). Spectrophotometry will be used to compare the optical density of each E. coli population and to create a ratio of red to optical density for the purpose of measuring the amount of red in each cell. This will tell us how effective the promoter is. Because we cannot work with the given repressor, we decided to alter temperature as a variable. We hypothesized that allowing the bacteria to incubate for one hour at a temperature of 42 degrees Celsius will act as an inducer for the specific promoter. We incubated samples for each of our experimental groups, which we separated into X1, X2, and X3, and then ran them in the spectrophotometer to compare the amount of fluorescence produced with the optic density of both the incubated and non-incubated groups. PCR was used to determine if the promoters were successfully cloned. PCR was done by adding PCR master mix (primer, DNA polymerase, dNTPs, and a buffer) to N and X1, X2, X3 groups. The bacteria provided the template for PCR. PCR results were run through gel electrophoresis to compare sizes. The added primer will be shorter (no longer than 60 nucleotides plus sticky ends) than the original primer in the N group that produced GFP. In moving forwards, we are currently looking into whether or not there is a gene that is similar to the lux R gene that is naturally expressed in E. coli that our promoter might be able to affect/function with. The bacteria in which this promoter is usually found also undergoes growth sue to density-based quorum sensing. To see if E. coli altered with this promoter can divide in the same way, we will take a dense cell colony, dilute it, and centrifuge it down. We will take an aliquot from the top of the centrifuged sample, which should theoretically contain some chemical that induces quorum sensing division. We will then spread that on another cell colony of the same dilution and see if it promotes growth.
Source
This promoter sequence came from an article published on NCBI: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444344/