Evaluation of a library of loxP variants with a wide range of recombination efficiencies by Cre

Sparse labeling of individual cells is an important approach in neuroscience and many other fields of research. Various methods have been developed to sparsely label only a small population of cells; however, there is no simple and reproducible strategy for managing the probability of sparse labeling at desired levels. Here, we aimed to develop a novel methodology based on the Cre-lox system to regulate sparseness at desired levels, and we purely analyzed cleavage efficiencies of loxP mutants by Cre. We hypothesized that mutations in the loxP sequence reduce the recognition efficiency by Cre, which enables the regulation of the sparseness level of gene expression. In this research, we mutagenized the loxP sequence and analyzed a library of loxP variants. We evaluated more than 1000 mutant loxP sequences, including mutants with reduced excision efficiencies by Cre ranging from 0.51% to 59%. This result suggests that these mutant loxP sequences can be useful in regulating the sparseness of genetic labeling at desired levels

大型藻類を用いたエタノール生産と造礁サンゴの環境ストレスからの保護を目的とした海洋資源の利用・保全技術に関する研究

京都大学0048新制・課程博士博士(農学)甲第22513号農博第2417号新制||農||1078(附属図書館)学位論文||R2||N5293(農学部図書室)京都大学大学院農学研究科応用生命科学専攻(主査)教授 植田 充美, 教授 渡邊 隆司, 教授 小川 順学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDFA

A Zeaxanthin-Producing Bacterium Isolated from the Algal Phycosphere Protects Coral Endosymbionts from Environmental Stress

サンゴの白化・絶滅を防御する天然の化合物を発見 --サンゴの共生バクテリアが放出する天然色素が褐虫藻のストレス耐性を上げる--. 京都大学プレスリリース. 2020-01-22.Reef-building corals form a complex consortium with photosynthetic algae in the family Symbiodiniaceae and bacteria, collectively termed the coral holobiont. These bacteria are hypothesized to be involved in the stress resistance of the coral holobiont, but their functional roles remain largely elusive. Here, we show that cultured Symbiodiniaceae algae isolated from the reef-building coral Galaxea fascicularis are associated with novel bacteria affiliated with the family Flavobacteriaceae. Antibiotic treatment eliminated the bacteria from cultured Symbiodiniaceae, resulting in a decreased maximum quantum yield of PSII (variable fluorescence divided by maximum fluorescence [Fv/Fm]) and an increased production of reactive oxygen species (ROS) under thermal and light stresses. We then isolated this bacterial strain, named GF1. GF1 inoculation in the antibiotic-treated Symbiodiniaceae cultures restored the Fv/Fm and reduced the ROS production. Furthermore, we found that GF1 produces the carotenoid zeaxanthin, which possesses potent antioxidant activity. Zeaxanthin supplementation to cultured Symbiodiniaceae ameliorated the Fv/Fm and ROS production, suggesting that GF1 mitigates thermal and light stresses in cultured Symbiodiniaceae via zeaxanthin production. These findings could advance our understanding of the roles of bacteria in Symbiodiniaceae and the coral holobiont, thereby contributing to the development of novel approaches toward coral protection through the use of symbiotic bacteria and their metabolites

The nucleotide bias at each position of the T7 promoter library.

<p>The nucleotide bias at each position of the T7 promoter library.</p

High-throughput evaluation of T7 promoter variants using biased randomization and DNA barcoding

<div><p>Cis-regulatory elements (CREs) are one of the important factors in controlling gene expression and elucidation of their roles has been attracting great interest. We have developed an improved method for analyzing a large variety of mutant CRE sequences in a simple and high-throughput manner. In our approach, mutant CREs with unique barcode sequences were obtained by biased randomization in a single PCR amplification. The original T7 promoter sequence was randomized by biased randomization, and the target number of base substitutions was set to be within the range of 0 to 5. The DNA library and subsequent transcribed RNA library were sequenced by next generation sequencers (NGS) to quantify transcriptional activity of each mutant. We succeeded in producing a randomized T7 promoter library with high coverage rate at each target number of base substitutions. In a single NGS analysis, we quantified the transcriptional activity of 7847 T7 promoter variants. We confirmed that the bases from −9 to −7 play an important role in the transcriptional activity of the T7 promoter. This information coincides with the previous researches and demonstrated the validity of our methodology. Furthermore, using an <i>in vitro</i> transcription/translation system, we found that transcriptional activities of these T7 variants were well correlated with the resultant protein abundance. We demonstrate that our method enables simple and high-throughput analysis of the effects of various CRE mutations on transcriptional regulation.</p></div

Strategy for construction of DNA and RNA samples.

<p>(A) Strategy for construction of DNA samples. DNA samples were constructed by two PCRs. The first PCR was performed to add universal sequence, a randomized T7 promoter, and a barcode sequence to the template. A second PCR was performed to amplify the DNA samples over 30 cycles. (B) Theoretical distribution of the number of base substitution for each randomization ratio. Blue line shows the retention rate at 90%. Orange line shows the retention rate at 70%. Black line shows the retention rate at 50%.</p

Coverage rate (%) of each base substitution group.

<p>Coverage rate (%) of each base substitution group.</p

Strategy for high-throughput evaluation of randomized T7 promoter sequences.

<p>(A) Construction of DNA fragments. DNA fragments possess a randomized T7 promoter and a random 16 bases (barcode sequence) upstream of the <i>ydaG</i> gene fragment. In addition, a universal sequence was added for NGS analysis (shown in Materials and methods). (B) NGS analysis of the DNA library. Through this analysis, the relationships between barcode sequences and T7 promoter variant sequences were clarified. In addition, the read number of each DNA sequence was counted. (C) Construction of a RNA library. RNA fragments were obtained by <i>in vitro</i> transcription of the DNA library. Transcribed RNA included barcode sequences. (D) NGS analysis of the RNA library. Through this analysis, the read count of each barcode was determined by NGS. (E) Integration of NGS data. By dividing RNA-derived barcode counts by the corresponding DNA counts, the transcriptional activity of each T7 promoter variant was calculated.</p

Correlation between the transcriptional activity and translational activity of <i>LacZ</i> gene with T7 promoter variant sequences.

<p>The plot illustrates the correlation between the transcriptional activity versus translational activity of LacZ production for four T7 promoter variants. Their relative transcriptional activities are 0.5, 0.2, 0.1 and 0.01. We used original T7 promoter sequence as a standard. In addition, we carried out an experiment with no DNA fragment as a negative control, and the transcriptional activity was set as zero. The translational activity was measured as the hydrolysis rate of 5-chloromethylfluorescein di-β-d-galactopyranoside (CMFDG). Each plot was averaged across three independent experiments. The error bars indicate standard errors of the means. RFU means relative fluorescence units.</p