Difference between revisions of "Team:Marburg/Improve"

There is always space for improvement, no matter how long you've been in the business.
-- Oscar de la Hoya

Cloning is the daily bread of molecular biologists. Almost all synbio projects start with weeks or months of cloning, a time which is the most frustrating and simultaneously least rewarding period of a project. Being able to distinguish between the native vector and the successfully assembled plasmid clearly while picking can save a lot of time and work. During the last decades, many approaches have been established to help distinguishing between false and correctly assembled plasmids. In our project, we created the Marburg Collection, a novel golden-gate-based cloning toolbox. This toolbox consists of LVL0 parts stored in a pSB1C3 derivat. As many plasmids had to be created, we desperately needed a simple, fast and cheap way to reliably select correct colonies even for inefficient clonings.

Here, we present the improvement of an iGEM-part for (a mostly faster,) easier and cheaper selection which suits our needs.

iGEM provides the universal acceptor plasmid BBa_P10500 for creating new PhytoBricks. This plasmid contained the lacZ-α part in the cloning sites for blue-white screening. The lacZ-gene encodes the β-galactosidase, which catalyzes the hydrolysis of the glycosidic bond of β-galactopyranosides like D-lactose. Blue-white screening is based on alpha-complementation, where the α- subunit (C-terminal section) and ω-subunit (N-terminal section) of the β-galactosidase (both non-functional peptides) are able to reconstitute a functional enzyme. By the addition of the two D-lactose Analogues Xgal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and IPTG (Isopropyl ß-D-1-thiogalactopyranoside) to the plates, colonies with successful cloning products thereby no lacZ-α will have the typical white colour while the unsuccessful ones precipitate the blue-coloured product resulting in blue colonies. For further reading: jana design/results