Part:BBa_K4216063
prmB promoter
Uri Alon Library of Promoter for E. coli
In 2006, Uri Alon screened over 2000 potential promoters for E.coli, to achieve this huge task, he and his team designed primers to flank intergenic regions that are longer than 40 bp. They then amplified the regions of interest: DNA located between two adjacent ORFs, the amplified region also extends 50–100 bp into each of the two ORFs. In the situation where no suitable primers were found, another extension is performed - but this time 300 bp into each of the ORFs. Each extracted region is then cloned into UA66 and pUA139, 2 vectors with containing a GFP reporter(Sigal_et_al._Nature_2006). To retrieve the sequence of each part, promoters used to extract the gene are bound to Escherichia coli str. K-12 substr. MG1655, forming the complete genome. The sequence is then extracted without the overhangs containing the enzyme restriction sites.
Here how we extract prmB promoter
Screening Validation and Characterisation
Materials and methods
The E. coli Promoter collection is delivered in 21 separate 96 well plates. Each well contains a strain stored in glycerol with a different promoter upstream of a Green Fluorecent Protein(GFP) coding sequence.
We used our custom 96 Well Plate Replicator to replicate the plates AZ_01 to AZ_05 of the promoter collection (a complete list of the library as well as the content of individual wells can be found here). We also prepared a plate with curated candidate promoters known to be implicated in the stress response, Redox sensing, Amino Acid synthesis and Ion Channel production in E. coli.
Cells were grown overnight in M9 minimal media (0.4% Glucose, no amino acids). this media was used to limit intefering substances found in rich media. In the morning the cells were diluted 1:100 in 96well plates with fresh media
We based our chosen electroshock amplitudes on those of a precedent paper [13] which produced different dynamics of membrane potential changes. 1 volt showed a strong un-sustained hyperpolarisation of the membrane, while 3 volts produced a less strong, sustained hyperpolarisation of the membrane.
Experience
To verify the ability of these promoters to respond to electrical signals as well as to characterise their activity, the previous 31 candidates were grown overnight in triplicates in a single 96-well plate in M9 minimal media (0.4% Glucose, no amino acids).
Cells were diluted 1:100 in 96-well plates with fresh media.
We repeated the 3 conditions used in the previous experiment and added 2 new conditions:
Condition B cells were exposed to an AC current of 1 volt, 100HZ, sine wave for 5seconds. Condition C cells were exposed to an AC current of 3 volts, 100HZ, sine wave for 20seconds. Condition D cells were exposed to an AC current of 1 volt, 100HZ, sine wave for 5 seconds every 15 minutes for 2 hours (cells were put back in the incubator in-between the shocks). Condition E cells were exposed to an AC current of 3 volts, 100HZ, sine wave for 20 seconds every 15 minutes for 2 hours (cells were put back in the incubator in-between the shocks).
After exposure to AC , cells from conditions A, B and C, were left to grow for 2 hours before measuring GFP expression for all conditions with a Flow cytometer.
We used 2 different selection methods to validate positive electrical response of promoters: A first selection was established for promoters which passed a certain threshold of expression (fold change < 0.8 and fold change > 1.2) in all triplicates.
In a second selection, the mean fluorescence of the triplicates was measured and filtered through the same threshold values to be validated as positive results.
This screening approach yielded 8 promoters with inducible activity and 11 promoters with repressive activity.
Results
Initial Screening
The following heatmaps show the fold change of GFP expression for each plates relative to their respective control. By hovering the mouse over the interactive plot, the reader can obtain the specific values of fold change. The first screening experiment yielded 31 candidates from a total of 545 promoter screened across 2 conditions.
Table 1 lists the 31 promoters that were identified in the preliminary screening. Data from plate AZ_05, condition B (1 volt, 5 seconds shock) were corrupted and could not be analysed. For the AZ_05 plate, data from condition C were collected the next day with the plate left in a -4*C freezer overnight. Promoters identified in this plate were discarded from the part collection as we esteemed these results too unreliable.
Out of these 31 candidates, 7 appeared in both screening conditions, 12 appeared in only one and 13 appeared in the single analysed condition for the AZ_05 plate.
AZ01
AZ02
AZ03
AZ04
AZ05
See here for interactive plot.
Validation and Characterisation
The following heatmaps show the fold change of GFP expression for each plates relative to their respective control plate in the 4 experimental conditions. By hovering the mouse over the interactive plot, the reader can obtain the specific values of fold change.
The Filter 1 refers to the first selection described in the material & methods section where each of the 3 triplicates have to pass the threshold to be considered valid. The Filter 2 refers to the second selection described in the material & methods section where the mean fluorescence of the triplicate have to pass the threshold to be considered valid
Figure 2A shows heatmaps of the Fold Change of GFP expression for all screened promoters, accross the 4 conditions and applying the 2 filters mentioned. Figure 2B shows the relative fold change of GFP expression in all promoters passing our each filtering across all conditions
Condition B : Exposure to 1volt peak to peak AC Current, 100HZ for 2.5 seconds Condition C : Exposure to 3volt peak to peak AC Current, 100HZ for 20 seconds Condition D : Exposure to 1volt peak to peak AC Current, 100HZ for 20 seconds every 15 minutes for 2 hours Condition E : Exposure to 3volt peak to peak AC Current, 100HZ for 20 seconds every 15 minutes for 2 hours
See here for more and interactive plot.
Table 2 lists the 19 promoters that passed our thresholds in one of the 2 filtering methods. Out of the 19 promoters, 4 appeared across all 4 conditions, 1 appeared in 3 condiction, 5 appeared in 2 conditions and 8 appeared in a single condition.
In all selected promoters, the effect of the electroshock is consistant accross all condition (if a promoter is repressed or induced in one condition, it is in all). We note 8 inducible promoters (with a fold change GFP fluorescence > 1.2 to their controls) and 11 repressible promtoers (with a fold change GFP fluorescence < 0.8 to their control)
The figure on below detail show the fold change fluorescence of the screened promoter compared to its control when exposed to specific electrical signals :
We mesure fold changes in the expression of the fluorescent protein between different shock conditions describe bellow:
Condition B : Exposure to 1volt peak to peak AC Current, 100HZ for 2.5 seconds Condition C : Exposure to 3volt peak to peak AC Current, 100HZ for 20 seconds Condition D : Exposure to 1volt peak to peak AC Current, 100HZ for 20 seconds every 15 minutes for 2 hours Condition E : Exposure to 3volt peak to peak AC Current, 100HZ for 20 seconds every 15 minutes for 2 hours
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 127
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 127
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 127
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 127
- 1000COMPATIBLE WITH RFC[1000]
| None |