Team:Freiburg/Cloning

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Main Project Modeling Applied Design Proof of Concept BioBricks Basic Part BioBrick Improvement Interlab Study

Safety Cloning HIF1A Knockdown Cell Culture Lab Book

Human Practice Integrated Human Practice Collaborations

Cloning

Cloning strategy

Our aim was to develop a cloning strategy providing us with plasmids that could be used for transient expression, to perform pretests of our CARTEL^TM AND gate, and as templates to generate transfer plasmids for lentiviral transduction. This resulted in the following cloning strategy.

Fig. 1: Summary of cloning strategy to generate plasmids suitable for transient transfection.
Shown are plasmids type one, that can already be used for promoter characterization pretests. Plasmids type two, that were built as subcloning plasmids to be assembled with plasmids type one into plasmids type three. Plasmids type four were generated by ligating four enhancer elements in succession, built by compatible end assembly, into plasmids type three.

As a first step, two plasmid types were generated. Plasmid type number one, assembled via Gibson Assembly, contained from 5’ end to 3’ direction one of the enhancers (HRE/CRE/pCTLA4), a minimal promoter to ensure better recruitment of the transcription machinery, one reporter protein (eCFP/SEAP/Luciferase) and a polyA signal sequence. The polyA signal was included to ensure better release of the transcribed mRNA from the transcription machinery and longer mRNA half-life (Hager et al., 2008). These plasmids were already suitable for pretests but were lacking a transfection control or a selection marker for lentiviral transduction.

The second plasmid type, also assembled via Gibson Assembly, contained from 5’ end to 3’ a constitutive promoter (CMV/SV40), a selection marker (mCherry/Zeocin/Neomycin) and a polyA signal sequence. These plasmids sole purpose was to be assembled with type one into type three plasmids. In a second step, plasmids type one and type two were assembled via a Gibson assembly into type three plasmids.

Considering that the enhancers one may only lead to very weak transactivation and therefore low reporter gene expression, plasmids containing an enhancer flanked by compatible restriction sites, suitable for compatible end assembly, were designed. To generate four enhancer elements in succession, compatible end assembly was performed twice. Additionally, two restriction sites, that were compatible with type three plasmids, were included, so that in a third step, type three plasmids and the four enhancer elements in succession could be combined via T4 ligation.

Fig. 2:Summary of cloning strategy to generate transfer plasmids for lentiviral transduction.
Plasmids type four were used as template to clone the constructs into transfer plasmids for lentiviral transduction.

In the fourth step, the assembled plasmids were cloned into a transfer plasmid via Gibson Assembly.

Cloning results

The strategy described above was designed to generate compatible plasmids for easy exchange, for e.g. reporter proteins or selection markers, providing us with our own cloning standard. During the time in the lab the following constructs were cloned.

Results

Cell Culture