Part:BBa_K774000:Design

Our hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular nitric oxide sensor that can be used in mammalian and bacterial cells interchangeably.

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 (Edinburgh 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 (Red text) and SpeI and PstI at the 3’-end (Blue text).

To provide additional restriction enzyme sites that may become useful during later cloning steps, BamHI, HindIII and NdeI (Green Text) 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.

References

Civerolo, K.L. and Dickerson, R.R. (1998) Nitric oxide soil emissions from tilled and untilled cornfields, Agricultural and Forest Meteorology, 90; 307-311

Davidson, E., (2012), Sources of Nitric Oxide and Nitrous Oxide following Wetting of Dry Soil, Soil Sci. Soc. Am. J. 56; 95–102

Lin H.Y., Bledsoe P.J., Stewart V., (2007), Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12▿, Journal of Bacteriology, 189: 7539 - 7548

Lipschultz, F., Zafiriou, O.C. Wofsy, S.C., Elroy, M.B., Valois, F.W. and Watson, S.W. (1981) Production of NO and N2O by soil nitrifying bacteria, Macmillan Journals, 294; 641-643

Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto, M., Stryhn, A., Ashmun, R.A., Shapiro, L.H., Arap, W. And Ruoslahti, E. (2000) Aminopeptidase N is a receptor for tumour-homing peptides and a target for inhibiting angiogenesis, The Journal of Cancer Research, 60; 722-727

Scott S.D., Joiner M.C., Marples B., (2002), Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy., Gene Therapy, 9: 1396-1402

Worthington J., Robson T., Scott S., Hirst, D., (2005), Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer, Gene Therapy, 12: 1417–1423

Xu, W., Liu, L.Z., Loizidou, M., Ahmed, M. And Charles, I.G. (2002) The role of nitric oxide in cancer, Cell Research, 12; 311-320