Difference between revisions of "Part:BBa K190015"

Revision as of 19:24, 25 October 2020

Arsenic Promoter (ArsR regulated)

Promoter arsRp is associated with the dimer of ArsR for the arsenic induced transcription of genes involved in arsenic efflux (arsR, arsB and arsC, which is present on the genome of Escherichia coli str. K-12 substrain MG1655). The sequence shows the typical -10 and -35 region of the promoter and can be found through the following [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link]. A second region, located at -41.5 from the transcription start site, is thought to bind dimeric ArsR. Upon binding of arsenic, the dimer dissociates and allows the RNA polymerase space to attach itself, and can also be found in the same [http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00239 link].

This set includes promoters that are sensitive to various metals. The promoters are typically regulated by a receptor protein that binds to the metal ion or complex.

Sequence and Features

Arsenic is an element less essential for the functioning of cells, and its survival. Low concentrations can have devastating effects, interfering with processes in the cell. Because of the similarity to phosphate, sometimes arsenate is mistaken for phosphate, which is how it is introduced into living organisms, including E. coli, by the phosphate uptake system. Other molecules such as As(III) can also be introduced into the cells by various membrane transporters. Cells have effective defenses to deal with elevated levels of arsenic variants, which are under the control of metal sensitive promoters. The transcription is mostly regulated by proteins binding to, or releasing from the promoter site, like ArsR.

• ArsR belongs to the ArsR/SmtB family of transcriptional regulators that respond to a variety of metals. ArsR has a helix-turn-helix motif for DNA binding, a metal-binding site, and a dimerization domain. In ArsR the inducer-binding site contains three cysteine residues that bind arsenite and antimonite specifically and with high affinity. Dimerization of ArsR is required for DNA binding and its ability to act as a transcriptional repressor. The dimer recognizes and binds to a 12-2-12 inverted repeat, but the binding of arsenic or antimonite to ArsR causes a conformational change in it, leading to dissociation from DNA and hence derepression (KEGG).

• ArsR negatively controls the expression of the genes involved in arsenical and antimonite metals resistance, whose expression is induced in the presence of these metals. The protein is autoregulated, because arsR is the first gene in the arsRBC operon that it regulates. Overexpression of ArsR in Escherichia coli has been used for removal of arsenite from contaminated water (KEGG).

(ArsR)2-DNA → ArsR-Ar + ArsR-Ar + DNA → Activation of transription

The presence of all genes and promoters on the chromosome of E. coli makes the use of the arsRp for induction of the GVP cluster relatively straith forward.

Fluorescence of resting cells

Upon induction of the ArsR promoter, expression of RFP increased, as seen in figure 1. From the enhanced fluorescence a value for the relative promoter units (RPU) was calculated according to the formula from Kelly 2009 (formula 9). Thereby an induction of 2.3 RPU was found, which was in consensus with the promoter activity found for arsenic metal sensitive promoter (used in expression of MTs) (personal communication, Dr. D. Wilcox). The arsenite uptake in ‘’E. coli’’ with J61002-BBa_K190015 over time was measured using the arsenite uptake assay, this was done upon incubation with 10µM NaAsO₂. This data was multiplied by the following ratio: As(III) uptake upon induction for 1hr with 100µM As(III) devided by As(III) uptake upon induction for with 10µM As(III). The increasing intracellular concentration is shown in figure 2.

Figure 1: Increase of fluorescence (RFP = 590nm) upon induction of the pArsR promoter with 100uM As(III). The data was a bit noisy, therefore a trendline was used to calculate the relative promoter units(RPU).

Figure 2: The increase of RFP over an increased intracellular As(III) concentration. The internal arsenic concentration upon induction of cells with 100uM As(III), was calculated by extrapolating the the As(III) uptake curve (incubated 10uM As(III) over 1hr) of E. coli with pArsR-RFP (in J61002). The trendline ((0.152465*x)/(1 + 0.121918*x)) was used to calculate the internal As concentration at the time point used for the fluorescence measurement.

Improvements

The BBa_K190015 promoter was not very effective in transcription repression as evidenced by using it in the construct BBa_K3380603. Therefore iGEM team Edinburgh 2020 decided to try and increase the transcription repression with ArsR transcription factor. We designed a promoter with the ArsR binding site placed after the promoter [BBa_K3380103]. The newly designed promoter was tested in a Cupriavidus Metallidurans cell free extract expressing iSpinach (BBa_K3380150) fluorescent RNA aptamer flanked by F30 tRNA scaffold (BBa_K3380101-BBa_K3380102). Figure 3 shows the effect of As (III) addition to the BBa_K3380600 construct under the BBa_K3380103 promoter.

Figure 3. C. Metallidurans cell-free extract containing the construct under the ArsR transcription factor repression expressing iSpinach Normalised Fluorescence over time in minutes.The T7 RNA polymerase and 7 μM As(III) were added at time= 0 min. The measurements were done in triplicates. The black triangle exhibits fluorescence of the construct BBa_K3380500 with 7 μM As(III) added. The green triangle exhibits fluorescence of the BBa_K3380600 construct with 7 μM As(III) and the blue dot exhibits fluorescence with the BBA_K3380600 construct with no metal addition. The details of the construct assembly and the exact concentrations of parts used can be found under the Experience page of the similar construct BBa_K3380500 [1]

Functional Parameters

Based on the experiments detailed above we conclude that the maximum promoter strength divided by the base promoter strength is about 2.3 for resting cells, also [http://2009.igem.org/Team:Groningen/Modelling/Characterization leading to a value for βRN·τR]: