Difference between revisions of "Part:BBa K1590009"
Franksargent (Talk | contribs) (→Functional Parameters) |
|||
| (6 intermediate revisions by 3 users not shown) | |||
| Line 10: | Line 10: | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | For semen detection our main target ligand is the polyamine spermidine which is found in relatively high concentrations in seminal fluid (5-15 mM). Spermidine is made from another polyamine called putrescine and is the precursor of spermine. Regulation of polyamine synthesis, degradation and transport is tightly controlled in bacteria. In E. coli, two of three identified transport systems are ABC transporters composed of a periplasmic binding protein, a pair of transmembrane proteins and a membrane protein possessing ATPase catalytic activity. Out of these three components, we were interested in investigating the periplasmic binding protein PotD which specifically binds spermidine. The fact that this protein is responsible for transportation of spermidine and lacking in enzymatic activity meant that it was an ideal candidate for use in FluID for semen detection | + | For semen detection our main target ligand is the polyamine spermidine which is found in relatively high concentrations in seminal fluid (5-15 mM). Spermidine is made from another polyamine called putrescine and is the precursor of spermine. Regulation of polyamine synthesis, degradation and transport is tightly controlled in bacteria. In E. coli, two of three identified transport systems are ABC transporters composed of a periplasmic binding protein, a pair of transmembrane proteins and a membrane protein possessing ATPase catalytic activity. Out of these three components, we were interested in investigating the periplasmic binding protein PotD which specifically binds spermidine. The fact that this protein is responsible for transportation of spermidine and lacking in enzymatic activity meant that it was an ideal candidate for use in FluID for semen detection. |
| − | + | ||
| − | + | ||
https://static.igem.org/mediawiki/2015/7/70/Semen-fig3.png | https://static.igem.org/mediawiki/2015/7/70/Semen-fig3.png | ||
<br> | <br> | ||
| − | Chromatogram showing the purification profile of the his-tagged bacterial protein, PotD. The fractions corresponding to the two peaks observed on the chromatograph were further analysed on SDS page gel. B) Coomassie Stain of the purified fractions (A12 + B12). 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100V and then stained. These were then western blotted to confirm the presence of our target protein PotD-His. | + | Figure 1: Chromatogram showing the purification profile of the his-tagged bacterial protein, PotD. The fractions corresponding to the two peaks observed on the chromatograph were further analysed on SDS page gel. B) Coomassie Stain of the purified fractions (A12 + B12). 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100V and then stained. These were then western blotted to confirm the presence of our target protein PotD-His. |
| + | https://static.igem.org/mediawiki/2015/a/ab/Semen-fig4.png | ||
| + | <br> | ||
| + | Figure 2: Samples from the SEC were ran on a 12.5% SDS-PAGE gel and then transferred to a nitrocellulose membrane and probed with an anti-His antibody. The band detected in the blot corresponds to the expected size of PotD of 37kDa. | ||
| + | ===Functional Parameters=== | ||
| + | <partinfo>BBa_K1590009 parameters</partinfo> | ||
| + | <i>E. coli</i> PotD is synthesised as a 348 amino acid residue precursor protein. After targeting to the periplasm a 28 amino acid N-terminal signal sequence is proteolytically removed. This results in a 320 amino acid residue periplasmic protein that acts as the periplasmic binding protein component of an ABC transporter. The mature portion of PotD contains eight tryptophan residues. These residues will fluoresce when exited by light at ~295 nm wavelength. This property can be used to study ligand binding by proteins. Typically, the relative fluorescence of a protein can be quenched in the presence of a ligand and careful ititration of the ligand can often give detailed information on the dissociation constant. | ||
| + | In this work, purified PotD (5 micro-Molar concentration) was observed by scanning fluorescence spectroscopy where an emission spectrum was collected after the tryptophans were excited by exposure to light at 295 nm wavelength. Different concentration of spermidine were then titrated into the protein solution and the quenching of the fluorescence spectrum observed. This suggests binding of the ligand to the protein and, given more time, this type of experiment can be used to estimate binding constants. | ||
| − | + | [[image:Dundee15PotDtrypto.jpg|left|200px|]] | |
| − | + | ||
| − | + | ||
Latest revision as of 15:45, 23 September 2015
Spermidine/putrescine-binding periplasmic protein
Escherichia coli PotD sequence, encoding Spermidine/putrescine-binding periplasmic protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 429
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 148
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
For semen detection our main target ligand is the polyamine spermidine which is found in relatively high concentrations in seminal fluid (5-15 mM). Spermidine is made from another polyamine called putrescine and is the precursor of spermine. Regulation of polyamine synthesis, degradation and transport is tightly controlled in bacteria. In E. coli, two of three identified transport systems are ABC transporters composed of a periplasmic binding protein, a pair of transmembrane proteins and a membrane protein possessing ATPase catalytic activity. Out of these three components, we were interested in investigating the periplasmic binding protein PotD which specifically binds spermidine. The fact that this protein is responsible for transportation of spermidine and lacking in enzymatic activity meant that it was an ideal candidate for use in FluID for semen detection.
Figure 1: Chromatogram showing the purification profile of the his-tagged bacterial protein, PotD. The fractions corresponding to the two peaks observed on the chromatograph were further analysed on SDS page gel. B) Coomassie Stain of the purified fractions (A12 + B12). 10 µl of each fraction was mixed with 10 µl of 2x Laemmli buffer and loaded onto an SDS gel which was ran for 1 hour at 100V and then stained. These were then western blotted to confirm the presence of our target protein PotD-His.
Figure 2: Samples from the SEC were ran on a 12.5% SDS-PAGE gel and then transferred to a nitrocellulose membrane and probed with an anti-His antibody. The band detected in the blot corresponds to the expected size of PotD of 37kDa.
Functional Parameters
E. coli PotD is synthesised as a 348 amino acid residue precursor protein. After targeting to the periplasm a 28 amino acid N-terminal signal sequence is proteolytically removed. This results in a 320 amino acid residue periplasmic protein that acts as the periplasmic binding protein component of an ABC transporter. The mature portion of PotD contains eight tryptophan residues. These residues will fluoresce when exited by light at ~295 nm wavelength. This property can be used to study ligand binding by proteins. Typically, the relative fluorescence of a protein can be quenched in the presence of a ligand and careful ititration of the ligand can often give detailed information on the dissociation constant.
In this work, purified PotD (5 micro-Molar concentration) was observed by scanning fluorescence spectroscopy where an emission spectrum was collected after the tryptophans were excited by exposure to light at 295 nm wavelength. Different concentration of spermidine were then titrated into the protein solution and the quenching of the fluorescence spectrum observed. This suggests binding of the ligand to the protein and, given more time, this type of experiment can be used to estimate binding constants.
