Difference between revisions of "Team:Dundee/Part Collection"
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<p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo.</p> | <p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo.</p> | ||
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Revision as of 19:42, 18 September 2015
Fingerprint Aging
Blood
Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo.
Figure 1 - This figure illustrates the relationship between fingertip, substrate and environmental conditions which collectively form a fingerprint and its constituents.
Part: BBa_K1590000 (hHBA)
FluID- Blood Detection
Coding sequence for Human haemoglobin A. Haemoglobin is the tetrameric protein molecule in red blood cells that carries oxygen. It is composed of four polypeptide chains, which in adults consist of two alpha (a) globin chains and two beta (b) globin chains. In blood plasma, haptoglobin binds free haemoglobin released from red blood cells, inhibiting its oxidative activity. The haptoglobin-hemoglobin complex can then be removed by the reticuloendothelial system which is a part of the immune system. Despite this, haemoglobin is still found free in the blood plasma at a concentration of up to 0.1g/l and this is what we hope to detect. .
Part: BBa_K1590001 (hHBB)
FluID- Blood Detection
Coding sequence for Human haemoglobin B. Haemoglobin is the tetrameric protein molecule in red blood cells that carries oxygen. It is composed of four polypeptide chains, which in adults consist of two alpha (a) globin chains and two beta (b) globin chains. In blood plasma, haptoglobin binds free haemoglobin released from red blood cells, inhibiting its oxidative activity. The haptoglobin-hemoglobin complex can then be removed by the reticuloendothelial system which is a part of the immune system. Despite this, haemoglobin is still found free in the blood plasma at a concentration of up to 0.1g/l and this is what we hope to detect.
Characterisation of haemoglobin beta following SEC (size exclusion chromatography). A) The sample of concentrated fractions containing haemoglobin beta from nickel affinity purification was loaded onto a SEC column and the protein was eluted. B) 10µl of each fraction corresponding to the two observed peaks was mixed with 10µl of laemmli buffer and loaded onto a SDS gel (12.5% acrylamide). The bands observable on the gel are in line with the expected size of haemoglobin beta - 16kDa. C) Western blotting was then carried out against an anti-his antibody to confirm the presence of hHHB- His.
Part: BBa_K1590002 (hHBN)
FluID - Blood Detection
Coding sequence for Human haptoglobin.Haemoglobin is the tetrameric protein molecule in red blood cells that carries oxygen. It is composed of four polypeptide chains, which in adults consist of two alpha (a) globin chains and two beta (b) globin chains. In blood plasma, haptoglobin binds free haemoglobin released from red blood cells, inhibiting its oxidative activity. The haptoglobin-hemoglobin complex can then be removed by the reticuloendothelial system which is a part of the immune system. Despite this, haemoglobin is still found free in the blood plasma at a concentration of up to 0.1g/l and this is what we hope to detect.
Characterization of Human haptoglobin following nickel affinity FPLC. A) Chromatogram showing the purification profile of the His-tagged Human haptoglobin. The fractions corresponding to the two peaks observed on the chromatograph were further analysed western blotting. B) 10µl of the fractions A8-A10 were mixed with 10µl of laemmli buffer and samples separated by SDS-PAGE (12.5% acrylamide) and transferred to a nitrocellulose membrane and probed with an anti-His antibody. The western blot shows successful production of hHHB – His (expected size 45kDa).
Part: BBa_K1590003 (PChr)
Chromate Detection
Chromate responsive promoter. The promoter PChr is suspected to be inducible by chromate.
Single colonies of JM110 + pSB1C3-Pchr-gfp (A) and MC1061 + pSB1C3-Pchr-gfp (B) were prepared for western blotting against GFP.
Part: BBa_K1590004 (ChrB)
Chromate Detection
Promoter sequence of chromate resistance operon of Ochrobactrum tritici 5bvl1. Regulator or Chromate responsive promoter. The protein encoded by this sequence is a putative chromate responsive repressor of PChr (BBa_K1590003).
Comparison of presence of GFP in MG1655 + BBa_K1058008, MG1655 + pSB1C3-Pchr-gfp (A) + pUniprom-chrB, and MG1655 + pSB1C3-Pchr-gfp (A) + pUniprom-chrB (opt) It was found that GFP was produced in the absence of chromate for both systems. This reason for these unexpected results could not be discerned, and further experiments are required to understand those. At this stage of the project the results indicate that ChrB might not be a repressor.
Part:BBa_K1590006 (LSS)
Fingerprint Aging
Lanosterol Synthase catalyses the reaction from 2,3-oxido-squalene (squalene epoxide) to Lanosterol. It is one of the enzymes in the enzymatic cascade that converts squalene to cholesterol through stepwise modifications of the substrate.
Detection of His-tagged LSS in whole cells of E.coli. Single colonies of E.coli strain M15 pREP4 harbouring LSS. Cells were used to inoculate 5ml of LB growth medium supplemented with 100ug/ml ampicillin and 50ug/ml Kanamycin. Once the OD600 reached 0.7 the cells were then induced with IPTG, as indicated. Cells were then grown for a further 4 hours at 37oC, 1ml aliquots were pelleted and cells reuspended in 100ul laemmli buffer and 20ul of samples were separated by SDS-PAGE (12% acrylamide) and transferred to nitrocellulose membrane and probed with anti-His antibody.
Part:BBa_K1590007 (Obp2A)
Nasal Mucus Detection
Human Odorant Binding Protein 2A is a 155 amino acid (excluding the signal peptide) lipocalin of relatively low molecular weight (19318 Daltons). Structurally it forms an 8 sheet beta barrel flanked by a c-terminal alpha helix that together forms an internal hydrophobic pore known as a calix. It is secreted by the olfactory epithelial cells of the nose where it lies in high abundance within nasal mucus. Its primary function in the human body is believed to be in the transport of hydrophobic odorant proteins across the otherwise impenetrable aqueous mucus layer to the olfactory receptors of the nose. Due to its high specificity and abundance within nasal mucus, OBP2A was selected as the protein for use in nasal mucus detection.
This figure illustrates the calculated miller’s activity of each control/sample and suggest that the OBP2A subunits aren’t interacting. This can be gauged from the sample on the far right of each graph. They suggest that OBP2A has a lower interaction than the negative/regulatory controls.
Part:BBa_K1590008 (LbpA)
Saliva Detection
Coding sequence for Lactoferrin Binding Protein A of Neisseria Meningitidis. This protein sequesters Iron for the host from Lactoferrin.
Part:BBa_K1590009 (PotD)
Semen Detection
Escherichia coli PotD sequence, encoding Spermidine/putrescine-binding periplasmic protein.
Characterization of PotD. A) Chromatogram showing the purification profile of the his-tagged Human HBN protein. 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 (12.5% acrylamide). C) Samples from the SEC were then western blotted using an anti-His antibody. The band detected in the blot corresponds to the expected size of PotD of 37kDa.
Part:BBa_K1590010 (Sbp)
Semen Detection
Murine Spermine Binding Protein Binds the carbohydrate spermine.
Successful production of GFP under the control of PChr
