Difference between revisions of "Part:BBa K774001:Design"
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The mammalian element of the hybrid promoter was produced by nine CArG elements (repeated elements of CC(A/T)(6)GG), a promoter previously used synthetically for nitric oxide synthase as a cancer therapy (Worthington ''et al.'', 2005) and developed from the EGR1 gene for early growth response protein 1 (Scott ''et al.'', 2002). The CArG aspect of the hybrid promoter has been known throughout the project as the mammalian promoter, or simply M. | The mammalian element of the hybrid promoter was produced by nine CArG elements (repeated elements of CC(A/T)(6)GG), a promoter previously used synthetically for nitric oxide synthase as a cancer therapy (Worthington ''et al.'', 2005) and developed from the EGR1 gene for early growth response protein 1 (Scott ''et al.'', 2002). The CArG aspect of the hybrid promoter has been known throughout the project as the mammalian promoter, or simply M. | ||
| − | =Design Notes== | + | ==Design Notes== |
| − | + | [[Image:Design notes BM-MB.JPG]] | |
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| + | The initial sequences requested for synthesis will be a hybrid of the PyeaR promoter and the E9-ns2 CArG promoter. Two versions will be synthesised, with the promoters altering their position in relation to the 5’-end of the sequence. | ||
| − | + | To facilitate cloning into the Biobricks, sequences contain EcoRI and XbaI sites at the 5’-end and SpeI and PstI at the 3’-end. | |
| − | + | To provide additional restriction enzyme sites that may become useful during later cloning steps, BamHI, HindIII and NdeI have been added between the 2 promoters. | |
| − | + | ||
| + | In the details below, sequences are shown as single-stranded DNA sequences. It is envisaged that any open reading frame (e.g. RFP or GFP) will be cloned “downstream” (i.e. at the 3’-end) of these promoter sequences. | ||
Revision as of 08:59, 26 September 2012
Six new biobricks produced and submitted to the registry with characterisation from fluorescence-based experiments.
Parts produced from this project:
Bacterial-Mammalian/B-M (PyeaR-CArG) Hybrid Promoter -- Mammalian-Bacterial/M-B (CArG-PyeaR) Hybrid Promoter -- B-M + eCFP -- B-M + RFP -- M-B + eCFP -- M-B + RFP
Our main project has resulted in the production of a hybrid bacterial and mammalian promoter optimised for induction by nitric oxide, nitrates and nitrites. We have ligated PyeaR, a known bacterial promoter and Part BBa_K216005 (Cambridge 2009) in the parts registry, with its mammalian counterpart, CArG. The resulting hybrid promoter has been synthesised in two orientations; PyeaR (bacterial, B) upstream of CArG (mammalian, M), nicknamed (B-M); and CArG upstream of PyeaR (M-B). These orientations were submitted to the parts registry as our first two biobricks.
Bacterial
After research around the subject and searching the parts registry a promoter known as PyeaR was decided upon as the bacterial element of the hybrid promoter; PyeaR is found in the yeaR/yoaG operon of Escherichia coli and is associated with induction by nitric oxide, nitrates and nitrites (Lin et al., 2007). PyeaR is repressed by two main repressors; Nar, which is regulated by nitrates and nitrites; and NsrR, which is regulated by nitric oxide (Figure 1.). One of the key elements of PyeaR is that it is not repressed in aerobic conditions, allowing for easier carrying out of experiments. The PyeaR aspect of the hybrid promoter has been known throughout the project as the bacterial promoter, or simply B.
After researching the parts registry we came across an existing PyeaR Biobrick that was created by the 2009 Edinburgh iGEM team. Part BBa_K216005 (Edinburgh 2009)
In order to begin to develop experiments to characterise the hybrid promoters + fluorescent proteins experiments were also carried out on a biobrick containing PyeaR + GFP (Part BBa_K381001, Bristol 2010).
A graphical representation of PyeaR. In the higher image PyeaR's activity is being repressed by both Nar and NsrR preventing transcription and the ultimate expression of a reporter. In the lower image nitrate/nitrite molecules have inhibited the activity of Nar, and nitric oxide has inhibited activity of NsrR, allowing for transcription to occur and subsequent expression of a reporter.
Mammalian
The mammalian element of the hybrid promoter was produced by nine CArG elements (repeated elements of CC(A/T)(6)GG), a promoter previously used synthetically for nitric oxide synthase as a cancer therapy (Worthington et al., 2005) and developed from the EGR1 gene for early growth response protein 1 (Scott et al., 2002). The CArG aspect of the hybrid promoter has been known throughout the project as the mammalian promoter, or simply M.
Design Notes
The initial sequences requested for synthesis will be a hybrid of the PyeaR promoter and the E9-ns2 CArG promoter. Two versions will be synthesised, with the promoters altering their position in relation to the 5’-end of the sequence.
To facilitate cloning into the Biobricks, sequences contain EcoRI and XbaI sites at the 5’-end and SpeI and PstI at the 3’-end.
To provide additional restriction enzyme sites that may become useful during later cloning steps, BamHI, HindIII and NdeI have been added between the 2 promoters.
In the details below, sequences are shown as single-stranded DNA sequences. It is envisaged that any open reading frame (e.g. RFP or GFP) will be cloned “downstream” (i.e. at the 3’-end) of these promoter sequences.