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Table of Contents Title 18.2. Crimes and Offenses Generally Chapter 7. Crimes Involving Health and Safety Article 7. Other Illegal Weapons 18.2-308.5:1. Manufacture, importation, sale, possession, transfer, or transportation of auto sears and trigger activators prohibited; penalty

"Auto sear" means a device, other than a trigger activator, designed for use in converting a semi-automatic firearm to shoot automatically more than one shot, without manual reloading, by a single function of the trigger.

"Trigger activator" means a device designed to allow a semi-automatic firearm to shoot more than one shot with a single pull of the trigger by harnessing the recoil energy of any semi-automatic firearm to which it is affixed so that the trigger resets and continues firing without additional physical manipulation of the trigger by the shooter.

D. Nothing in this section shall be construed to prohibit a person from manufacturing, importing, selling, offering for sale, possessing, receiving, transferring, or transporting any item for which such person is in compliance with the National Firearms Act (26 U.S.C. 5801 et seq.).

Round Robin Item Use: The Autonomous Activator will progressively cycle through available items.

Randomly Use Items: Items in the inventory will be used randomly.

First Slot Only: Only the item in the top left box will be used.

Anything can be placed in the nine available slots, allowing the player to place, break, or attack anything in the block directly in front of the Autonomous Activator. Useful for automating tasks such as killing mobs, and planting and harvest crops. It does not require external power however it can be set to respond to a redstone signal.

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A common problem of the Y2H system is the autoactivation of Y2H-inducible reporter genes14, which can result in a large number of false positives. This occurs when either DNA binding domain (BD) or activation domain (AD) activates transcription of Y2H reporter genes irrespective of the presence of any PPI. Three types of autoactivators (AAs) can be observed: (i) transcription factors that natively contain a functional AD that is active when fused to BD, (ii) proteins that are not transcription factors but contain a cryptic AD, and (iii) proteins that are not transcription factors, that contain one or more cryptic ADs that are only functional as truncated fragments.

The conditioned cells were collected by centrifugation, washed once by 50 mL ice-cold electroporation buffer, and re-suspended in 100 to 200 L electroporation buffer to reach a final volume of 1 mL. The electrocompetent cells were now ready to use and kept on ice until electroporation.

In our pursuit to develop a genetic tool that can remove auto-activators from the yeast two-hybrid assay, we had to establish two components of the tool. First, we needed a negative selection marker that can inhibit the growth or kill the yeast cells. Second, we needed a means to conditionally activate the negative selection marker only when an auto-activator was present in the yeast cell.

We synthesized pGAL2-URA3 fragment and cloned it into pRS305K plasmid, which can be used to integrate the fragment into Leu2 chromosomal locus and select for the transformation by using kanamycin (Fig. 1A). The sequence of the vector is available as Supplemental Data 1. We have integrated the fragment into the genomes of yeast strains used in CrY2H-seq13, which use a Cre recombinase to fuse in vivo the coding sequences of two interacting proteins, allowing to identify these interactions with next-generation DNA sequencing.

We hypothesize that by adding FOA to the media before the mating, growth of haploid yeast cells that express URA3 should be inhibited (Fig. 1D), due to the accumulation of the toxic FU in the yeast cells. This should reduce or eliminate the number of auto-activators in our library population, and subsequently reduce the number of false-positive hits when used for mating in a yeast two-hybrid assay.

To test our AA-removal method, we set to identify genes that are AAs in the activation domain (AD) and the DNA binding domain (BD) libraries. We chose Marchantia polymorpha as a source for AAs, as it is an emerging model for early diverging plants, has a low number of genes, and minimal genetic redundancy17.

However, when SC-Leu media was supplemented with 0.2% FOA, AAs did not show observable growth after 1 day, while the EV showed a slower growth (Fig. 2C, right). On day 2, AAs showed minor growth, and their OD was 5.3 times lower compared to EV, indicating that the growth of AAs is penalized in FOA. On day 3, auto-activators and EV showed similar growth. This indicates that FOA can inhibit the growth of AAs, but the negative selection loses its potency after day 2 in liquid media. Consequently, we recommend to limit the FOA treatment to two days.

Taken together, based on results from this and previous sections, we can conclude that the negative selection using pGAL2-URA3 and FOA screening works well in liquid and solid media, preventing the growth of AAs. Our method successfully removed all auto-activators from a culture containing 83% AAs.

Since AAs are a large problem in Y2H screens and are difficult to remove by screening when performing a large-scale assay where millions of interactions are tested, we have developed a method that removes AAs by using conditional negative selection with URA3 gene, driven by pGAL2 promoter. The pGAL2 promoter activates URA3 only in the presence of an AA or a genuine PPI, allowing URA3 to convert 5-FOA into a toxic FU. When applied to haploid AD and BD cultures, the method has thus potential to selectively minimize or outright eliminate yeast cells harboring AAs.

The previous study has shown that AAs comprise 16% of baits13, which in turn comprised 95% of detected interactions in their large-scale CrY2H assay. In our study, we observed that BD and AD libraries contained 0.13% and 0.0029% AAs, respectively (Fig. 2A). We speculate that the higher number of AAs observed in the CrY2H study is because the authors focused on transcription factors13, which are likely to contain bona fide activation domains. Conversely, since we have studied all coding sequences in Marchantia polymorpha, the proportion of transcription factors containing activation domains should be lower, explaining the lower number of observed AAs. Despite the lower numbers in Marchantia, removal of AAs is still a worthwhile pursuit as it will result in a higher number of detectable true positive interactions.

We observed that the FOA treatment effectively suppresses the growth of AAs on solid media (Fig. 2B and 3B), and liquid media (Fig. 2C). However, the efficacy of the assay in liquid media drops after three days of incubation, as the growth of AAs equals that of the non-AAs (Fig. 2C, right panel). This indicates that our approach is only effective in the first two days. While the molecular basis for the loss of efficacy is unclear, we speculate that the unwanted growth could be caused by spontaneous mutations of the pGAL2-URA3, which either inhibit the activation of URA3 or inactivate the enzyme. Further work should attempt to introduce another copy of pGAL2-URA3 into the genome to provide a genetic backup for the assay.

We were able to remove all AAs from the panel of 48 colonies, indicating that despite the loss of efficacy after two days of cultivation in FOA, the assay is still able to deplete the AAs from a yeast culture (Fig. 3B). Furthermore, since the URA3 gene is also activated by true interactions (Fig. 2B), including pGAL2-URA3 into Y2H assays will have a valuable dual role of removing AAs and selecting for true PPIs.

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