Transposon-Based Directional Polypeptide Truncation
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Solution Overview
Problem
Current methods for generating deletions in nucleic acids are time-consuming and inefficient, often requiring cloning of the nucleic acid sequence into a vector, and lack the ability to produce large quantities of deletions in a random fashion, limiting the discovery of unexpected results.
Innovation Solution
The method involves inserting a transposon into a target sequence, amplifying the transposon-containing sequence, and transforming it into a host cell to produce unidirectionally or bidirectionally truncated polypeptides, allowing for random and efficient generation of deletions without the need for initial cloning, using a transposon with a selectable marker and origin of replication for replication and selection in the host cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current techniques for generating deletions in nucleic acids are used, then deletions can be generated, but the process is time-consuming and inefficient
Solution Approach 1:
The patent extracts the essential function of deletion generation by using transposons that naturally insert into DNA at random locations. The transposon carries a selectable marker and origin of replication, allowing direct generation of truncated polypeptides without requiring prior cloning into vectors. This extraction of the core function eliminates time-consuming cloning steps while maintaining deletion generation capability.
Solution Approach 2:
The transposon is pre-equipped with a selectable marker and origin of replication before insertion into the target sequence. This preliminary preparation allows the transposon to immediately enable selection and replication in host cells upon insertion, eliminating the need for subsequent cloning and selection steps that would otherwise be required.
2Ease of manufacture
If current techniques are used, then deletions can be generated, but cloning of the nucleic acid sequence into a vector is required
Solution Approach 1:
The patent removes the vector cloning step from the deletion generation process by using transposons that can directly insert into linear or circular DNA molecules. The transposon system eliminates the need for intermediate vector construction, making the process simpler and more direct.
Solution Approach 2:
The transposon acts as an intermediary element that carries both the selectable marker and origin of replication. This intermediary function allows direct transformation into host cells without requiring vector intermediaries, simplifying the overall process while maintaining all necessary functions.
3Quantity of substance
If current techniques are used, then deletions can be generated, but large quantities of deletions cannot be produced in a random fashion
Solution Approach 1:
The transposon is pre-designed with random insertion capability and carries all necessary elements (selectable marker, origin of replication) for immediate functionality. This preliminary configuration enables high-throughput generation of diverse truncated polypeptides in random fashion, producing large quantities of different deletion variants simultaneously.
Solution Approach 2:
The patent utilizes the random insertion parameter of transposons to generate diverse truncated polypeptides. By controlling transposon insertion into different locations within the target sequence, the system can produce a wide variety of deletion lengths and positions, increasing both the quantity and diversity of truncated polypeptides generated efficiently.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables the rapid and efficient production of truncated polypeptides, facilitating research and therapeutic applications by reducing time and increasing the likelihood of obtaining unexpected and valuable results through random deletion processes.
Implementation Method 1
inserting a transposon into a target sequence to generate a transposon-containing target sequence
Implementation Method 2
amplifying the transposon-containing target sequence to generate an amplification product
Implementation Method 3
transforming the amplification product into a host cell
Implementation Method 4
the circular amplification product is replicated using the origin of replication from the transposon
Implementation Method 5
the selectable marker encoded by the transposon is expressed
Data Source
AI summary
Methods, compositions and kits are disclosed for obtaining directionally truncated polypeptides by inserting a transposon. Preferably the transposon comprises a selectable marker and an ori, and optionally a promoter, a ribosome binding site and a translation start codon, into a target sequence in vitro or in vivo. Amplification products, varying in length depending on the transposon insertion site, are obtained using one primer that anneals to the target sequence and a second primer that anneals to the transposon. Amplification products are ligated to circular dsDNA, transformed into host cells, and individual colonies, each containing a directionally truncated clone of the target sequence, are obtained by plating on medium for which the selectable marker encodes resistance. Directionally truncated polypeptides encoded by the target sequence are obtained in vivo by inducing an RNAP in the host cells that uses the promoter or, in vitro by cell-free transcription and translation.