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From 2010.igem.org

• BBa_K381001 • Promoter • Reporters • PyeaR + LacZ construct • LacZ •

LacZ

The motivation for the use of the LacZ reporter sequence in our experiments stems from our initial attempts to characterise the PyeaR promoter sequence using the [http://partsregistry.org/Part:BBa_K216009 BBa_K216009] part created by the Edinburgh team for the 2009 competition.

LacZ is the reporter sequence used to assay promoter sensitivity by way of a β-Galactosidase assay - it encodes the enzyme β-Galactosidase. The molecular basis of this experiment is the cleavage of the compound o-nitrophenyl-β-D-galactoside (ONPG), an analogue of lactose (the primary substrate of β-Galactosidase), to galactose and o-nitrophenol. The latter is a yellow product with an easily measurable absorbance at 420 nm. When ONPG is added to a reaction mix in excess of the β-Galactosidase enzyme, the production of o-nitrophenol over time is a direct indicator of the activity of the enzyme. Thus if LacZ is regulated by a specific promoter, the activity of the enzyme is directly proportional to the activity of the promoter.

From the data collected from this asasy, a value for the activity of a promoter can be derived using the following equation:

(A₄₂₀ gives a value for o-nitrophenol concentration; A₅₅₀ gives a value for cell debris scattering (the procedure of the β-Galactosidase assay requires cells to be broken apart); 0.1 is the volume of cell lysate used in the assay, and; A₆₀₀ gives the cell density before lysis).

Returning to agriColi, the further characterisation of PyeaR was essential to inform our work and to give us a clear understanding of the activity of PyeaR in a range applicable to the nitrate levels in soil. However, after several failed attempts with [http://partsregistry.org/Part:BBa_K216009 BBa_K216009] it was discovered that the title for this biobrick on the registry is misleading - [http://partsregistry.org/Part:BBa_K216009 BBa_K216009] only contains LacZ' or LacZα (depending on which nomenclature you choose to apply), rather than the full copy of LacZ. This requires the second part of lacZ, known as LacZΩ to be present on the bacterial genome for the assay to function correctly.

This is more appropriate for the technique of blue-white screening than for β-Galactosidase assays. For a start, the activity of the promoter may cause greater production of LacZα than LacZΩ, yet the activity of β-Galactosidase will be limited by the expression of LacZΩ and hence give a false limit to the activity of the promoter being assayed. Coupled with the stress of adding extra reagents such as IPTG to the mix, this results in a procedure that is not appropriate for complete characterisation of the biobrick. With this in mind, the team set out to create a new biobrick using the promoter PyeaR and a full functional copy of LacZ.

Despite our best efforts and two attempts at forming the new construct, none of our efforts were successful, and with time constraints in mind we returned to [http://partsregistry.org/Part:BBa_K216009 BBa_K216009] in order to get the best data we could from the materials we had. After several more attempts we finally arrived at a set of reproducible data and at this point called a halt to the lab work. The results obtained from the numerous repeats can be viewed on the β-Galactosidase assay page in this wiki.