Faster Bacterial Gene Cloning Using the Brick into the Gateway (BiG) Protocol.

Cloning systems like Gateway and Golden Gate/Braid are known because of their efficiency and accuracy. While the main drawback of Gateway is the expensive cost of the enzymes used in its two-step (LR and BP) reaction, Golden Gate requires non-reusable components due to their specific restriction sites. We present the Brick into the Gateway (BiG) protocol as a new cloning strategy, faster and more economic method that combines (i) reusable modules or bricks assembled by the GoldenBraid approach, and (ii) Gateway LR reactions [recombination of attachment sites: attL (L from left) and attR (R from right)] avoiding the BP reaction [recombination of attachment sites: attP (P from phage) and attB (B from bacteria)] usually necessary in the Gateway cloning. The starting point is to perform a PCR reaction to add type IIS restriction sites into DNA fragments generating specific fusion sites. Then, this PCR product is used to design GoldenBraid bricks, including the attL Gateway recombination sites. Using the Golden Gate method, these bricks are assembled to produce an attL1 -gene of interest- attL2 fragment, which is integrated into a compatible vector producing a Gateway entry vector. Finally, the fragment containing the target gene is recombined by LR reaction into the Gateway destination vector. This protocol was validated in: Plasmid (2022), DOI: 10.1016/j.plasmid.2022.102630 Graphical abstract.

Citogenética comparativa de Physalis peruviana en tres poblaciones cultivadas de Cajamarca, Perú / Comparative cytogenetics of Physalis peruviana from three cultivated populations from Cajamarca, Peru

Resumen En este trabajo se caracteriza y compara citogenéticamente Physalis peruviana "aguaymanto" de poblaciones cultivadas de la región Cajamarca: San Pablo, Celendín y Cajabamba. El número cromosómico más frecuente en las tres poblaciones fue 2n = 4x = 48 con frecuencias de 60, 50 y 34% en las poblaciones de San Pablo, Celendín y Cajabamba respectivamente. En menor frecuencia se encontró casos de aneuploidía somática. Los resultados permitieron distinguir cada población a partir de su fórmula cariotípica 17m+4sm+3t, 24m y 20m+2sm+2t que identifican a San Pablo, Celendín y Cajabamba respectivamente. Los cariotipos fueron caracterizados por la longitud total del complemento haploide (HCL) y los índices de simetría (S%), asimetría (A) y asimetría intra e intercromosómica (A1 y A2). El mayor valor de HCL se describió en San Pablo. Celendín presentó el mayor grado de simetría (S%=53.226 y A= 0.177), mientras que los cariotipos de San Pablo y Cajabamba fueron descritos como los más asimétricos. Se concluye que la condición más frecuente es la tetraploidía; aunque se evidencia diferente morfología cromosómica entre los cariotipos de las tres poblaciones.

Abstract In this work, we characterized cytogenetically Physalis peruviana "aguaymanto" and cultivated populations of the Cajamarca region: San Pablo, Celendín and Cajabamba are compared. The most frequent chromosomal number in the three populations was 2n = 4x = 48 with frequencies of 60, 50 and 34% in San Pablo, Celendín and Cajabamba respectively. Few cases of somatic aneuploidy were found. Our results let distinguish the populations by its karyotypic formula 17m + 4sm + 3t, 24m and 20m + 2sm + 2t (San Pablo, Celendín and Cajabamba respectively). Karyotypes were characterized by the total length of the haploid complement (HCL) and the indices of symmetry (S%), asymmetry (A) and intra and interchromosomal asymmetry (A1 and A2). The highest value of HCL was described in San Pablo. Celendín presented the highest degree of symmetry (S% = 53.226 and A = 0.177), while the karyotypes of San Pablo and Cajabamba were described as the most asymmetric. We concluded that the tetraploidy is most frequent condition; although there is evidence of different chromosomal morphology between the three populations.

Diversidad genética de tres poblaciones de Physalis peruviana a partir del fraccionamiento y patrón electroforético de proteínas de reserva seminal / Genetic diversity in three populations of Physalis peruvianausing fractionation and electrophoretic patterns of seed storage protein

En el presente trabajo, es estudiada la diversidad genética de tres poblaciones atribuidas a ecotipos de aguaymanto, Physalis peruaviana. Las tres poblaciones eran atribuidas a los ecotipos Agroandino (provincia de San Pablo), Celendino (provincia de Celendín) y Cajabamba (provincia de Cajabamba) del departamento de Cajamarca. Se realizó la cuantificación proteica y evaluó el polimorfismo de las proteínas de reserva seminal (SSPs) mediante electroforesis en gel de poliacrilamida denaturante (SDS-PAGE). Además, se identificaron características bioquímicas de las proteínas seminales en esta especie. No se hallaron diferencias entre las tres poblaciones basados en la cuantificación proteica. Las globulinas (82.4%) fueron la fracción mayoritaria seguida por las albuminas (13.9%), glutelinas (3.7%) y prolaminas (0.7%). Sólo las albuminas mostraron polimorfismo, hallándose 21 proteínas entre ~ 6.5 a ~45 kDa y tres perfiles electroforéticos diferentes, los cuales fueron compartidos entre las poblaciones. Se identificaron las leguminas y vicilinas en la fracción globulina. Las glutelinas mostraron proteínas de mismo peso molecular (PM) a las leguminas; y las prolaminas sólo una banda de bajo PM. La población de San Pablo fue completamente homogénea a diferencia de la población de Cajabamba que mostró la mayor diversidad genética seguida de Celendín. No fue posible diferenciar las poblaciones designadas como ecotipos Agroandino, Cajabamba y Celendino basados en el análisis de proteínas seminales.

The genetic diversity of three populations designated as ecotypes of golden berry (Physalis peruaviana) is studied using protein quantification and polymorphism of seed storage proteins (SSPs) by denaturating polyacrylamide gel electrophoresis (SDS-PAGE). As well, biochemical characteristics of seed proteins were identified. The populations were from San Pablo province (Agroandino ecotype), Celendín province (Celendino ecotype) and Cajabamba province (Cajabamba ecotype), all from Cajamarca Department. There was not difference among the three populations based on protein quantification. Globulins (82.4%) were the majority fraction followed for albumins (13.9%), glutelins (3.7%) and prolamins (0.7%). Only albumins showed polymorphism, showing 21 proteins between ~6.5 to ~45 kDa and three different electrophoretic profiles, which were share among the three populations. Legumins and vicilins were identified in globulin fraction. Glutelins showed proteins of same molecular weight (MW) to legumins; and prolamins only a band of low MW. San Pablo province population (Agroandino ecotype) was completely uniform, while Cajabamba population showed higher genetic diversity followed by Celendin population. Our results shows that, based on seed proteins analyses is not possible to distinguish the three populations designated as Agroandino, Cajabamba and Celendino ecotypes.