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I'm writing my master's thesis proposal, and I need to improve the solution-behavior of some truncated protein domains which have previously shown poor solubility when expressed in E. coli.

To prevent this unwanted aggregation, one experiment I will perform will be to alter the domain boundaries. I will create gene constructs where each clone will have an A domain of a certain length and domain border (see below).

Note: insert size is ~1.3kb

My question: Is Gateway cloning better than restriction/ligase methods for this? If i use restriction/ligase methods to construct vectors for transformation, then my choice for domain boundaries will be determined by restriction sites. Howevever, with a system like Gateway cloning, I can define boundary limits to bp resolution by simply desining an appropriate primer.

Is my reasoning here correct? I need to justify my choises for experimental design to researchers, so want to make sure I'm being efficient and logical.

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  • $\begingroup$ Take a look at this. $\endgroup$
    – user137
    May 20, 2016 at 11:19

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Yes. In principle you will have a vastly increased ability to customize the boundaries of each protein domain with a primer and PCR based approach. It is not obvious to me that the Gateway Cloning System is required, or necessarily the best choice for this approach, but that is likely subjective.

Are you familiar with, or aware of, the efforts of the SGC (Structural Genomics Consortium)? My understanding (now somewhat outdated) is that they were going to design/use a semi-automated way of implementing the strategy you discuss (domain trimming) so that they could us a high-throughput approach to define crystallization conditions for their targets. Perhaps you could adapt their method(s) to your protein of interest?

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