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.

Figure 1: Plasmidmap of BBa_K2560002
Our improved part BBa_K2560002 is a derivate of the BBa_P10500 containing sfGFP drop out.

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, click here.
However, this kind of selection requires the use of specific strains which possess the lacZ-ω but neither the native lacZ nor the lacZ-α fragment. As an example, the E. coli NEB Turbo strain which is routinely used for cloning harbors a lacZ without the N-terminal portion. As we wanted to establish Vibrio natriegens as a chassis for cloning, we investigated if the wild type strain is compatible with blue-white screening. Unfortunately, cloning using BBa_P10500 in V. natriegens shows no difference between colonies containing the lacZ-α dropout and those without. For further reading about the lac operon of V. natriegens, click here.

To overcome this limitation and to enable fast and reliable cloning with V. natriegens, we decided to establish a new visualization method. Our improved part, namely BBa_K2560002, is a derivative of the iGEM BBa_P10500 containing a sfGFP dropout as fluorescent selection marker.
In the pictures 2, you can see the wild type V. natriegens in comparison with the iGEM BBa_P10500 forming white colonies as well as bright green colonies containing the improved part BBa_K2560002. No differences between the wild type and the BBa_P10500 containing colonies are noticable. On the contrary, our new BBa_K2560002 part leads to a strong visual distinction to colonies which do not possess the sfGFP. In this way, we created a part for universal LVL0 cloning with a improved selection without the need of additional supplements like Xgal or IPTG.

Our part not only suits for V. natriegens but is convenient for the frequently used cloning host E. coli. As it can be seen in the picture 3, E. coli containing the sfGFP possess a considerable strong green colour even without the use of UV light and is just or even more distinguishable from the wild type as the lacZ containing blue colonies. By using our improved part BBa_K2560002 instead of the iGEM part BBa_P10500 work and money for the addition of the required supplements can be saved and the risk of not functional plates for selection is decreased. In our experiments, we were using 40 μg per Liter Xgal and 0,5 mM IPTG. Calculating with current prices, 100 plates supplemented with Xgal and IPTG costs about 80 dollar.
Figure 2: V. natriegens
A: Colonies of wild type, B: white colonies containing BBa_P10500 and C: colonies with BBa_K2560002 resulting in green colonies.
Figure 3: E. coli
A: Colonies of wild type, B: blue colonies containing BBa_P10500 and C: colonies with BBa_K2560002 resulting in green colonies.

By using our improved part for cloning, the detection of successfully ligated clones from non is feasible for strains which are not compatible with blue-white screening and therefor becomes more universal, faster and cheaper than before.

B. Marchal