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Will Shaw

@willshaw.bsky.social

325 followers 198 following 17 posts

Postdoc in Mo Khalil’s lab at Boston University • synthetic biology • genome engineering • plants • yeast

Will Shaw @willshaw.bsky.social · 11/06/2026

By considering transformation outcomes holistically, and balancing the key criteria for successful transgenesis, we believe the T1 vectors will help reduce a major bottleneck in Arabidopsis research. 14/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

What to expect with the T1 vectors: A single 12 cm selection plate with 5,000 seeds yields ~25 transformants. Using low-effort genotyping, we mapped around one-third as single-copy insertions free from vector backbone sequence. 13/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

We combined these advanced to form the T1 vector series, an Arabidopsis-optimised T-DNA vector system. The T1 vectors enable clean, single-copy, and readily mappable transgene integration with predictable expression in the first generation after transformation. 12/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

We didn’t stop there! We also developed a simple genomic mapping method using inverse PCR, NsiI restriction sites, and iPCR barcodes next to each T-DNA border. Using this we are able to easily map clean, single-copy T-DNA insertions in Arabidopsis. 11/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

The Arabidopsis EF1A3 promoter worked well for selection marker expression while avoiding unwanted enhancer activity. This supports more predictable cell-type-specific and conditional transgene expression. 10/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

We also tackled a common issue with T-DNA vectors: the 35S promoter often used for selection markers has strong enhancer activity that can drive ectopic expression of adjacent transgenes. To avoid this, we screened Arabidopsis promoters for better alternatives. 9/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

As shown previously, this transformation boost also works in Arabidopsis. However, it also caused a large increase in T-DNA copy number. Here, more plants are not necessarily better, because more screening is needed to find stable, single-copy transgenic lines. 8/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

We also tested the effects of increasing T-DNA vector copy number in Agrobacterium, inspired by the excellent work of Szarzanowicz et al. Using a high-copy pVS1 replicon mutation, we confirmed improved transient transformation in Nicotiana. 7/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

We designed three T-DNA vectors with low, medium, and high transformation efficiency. The medium-efficiency design, RB-Mid, provided the best balance: good transformation efficiency, lower copy number, and reduced vector backbone transfer. 6/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

Using this reporter, we screened T-DNA border variants in a conventional vector similar to pCAMBIA. Altering the virulence-enhancing overdrive sequence revealed major differences in transformation efficiency and the fraction of plants expressing RUBY. 5/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

We developed a visual reporter for screening of predominantly single-copy insertions, based on RUBY transgene silencing seen in plants with more than ~1 T-DNA. Combined with simple genotyping, this allowed us to assess transformation outcomes beyond efficiency at scale. 4/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

Researchers often focus on improving transformation efficiency, but other outcomes are equally if not more important. Transgene copy number and expression stability all determine whether a transformant is useful. Screening these properties is laborious and time-consuming. 3/16

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Will Shaw @willshaw.bsky.social · 11/06/2026

In this paper, we set out to address one of the major bottlenecks in plant science - challenges associated with transformation. T-DNA integration is often multi-copy and structurally complex. This leads to a lot of variability, and often transgene silencing. 2/16

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