Cloning objectives in pydna

[BjornFJohansson]

A cloning project is a series of cloning steps originating in a set of DNA molecules (sequences) resulting in another set.

Pydna can can capture the minute details of the cloning process, but at this point not the final objective of the project.

The objective of cloning is often (but not always) the expression of a protein. This protein can be a mutant like the result of directed protein engineering. It can also be a fusion protein, like the N- or C- terminal GFP fusions created for visualization.

Graphical tools like snapgene or benchling allows for the user to manually
inspect sequences with highlighted features to allow for low-throughput
verification that the final goal was achieved. However, this is as error prone as the ad-hoc cut-and-paste cloning itself.

Several types of minor mistakes that are hard to detect can cause severe
problems. Examples of such problems are:

• Incomplete or too large protein due to a mislocated forward and/or reverse primer
• Frame shifts due to forward primer design error.
• Internal stop codons, sometimes left in fusion proteins.
• Upstream start codons.
• Primer introduces unintended mutations (single-base errors, codon changes)
• High GC or secondary structure in mRNA reduces expression efficiency.
• Repetitive sequences triggering recombination or deletion in E. coli or yeast.
• Fusion junction produces low-complexity regions or unwanted protease cleavage sites.
• Accidental introduction of a rare codons at the fusion boundary.
• Missing or duplicated ribosome binding site (RBS/Kozak sequence).

Each of these problems might not be caught at the design level, as the result may seem correct.

Solution(s):

Establish an objective as early as possible. This objective could be a string (below)
containing one or more protein sequences.

The objective below pertains to a gfp fusion experiment.

The objective was to express a C-terminal GFP fusion between the JEN1 transporter
and GFP.

• JEN1 protein sequence (616 aa) MSSSITD...EEHIETV
• GFP (237 aa) SKGEEL...MDELYK

How should pydna help to show if the goal has been attained?

TBD