A split GFP system to enhance spatial and temporal sensitivity of translating ribosome affinity purification (TRAP)

SummaryTranslating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity purification of ribosomes and the mRNAs that they are translating. These actively translated mRNAs (translatome) can be interrogated by qPCR or RNAseq. Condition- or cell-specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell-specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: one is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split GFP; the second is controlled by a stimulus-specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi-molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre-existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant-pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies.SignificanceTranslating ribosome affinity purification (TRAP) has been modified to allow with increased sensitivity the isolation of RNA from sets of cells in which the activity of condition/cell-specific promoters is weak, ribosome turnover is low, or cells whose nature is ephemeral. Based on the use of a split linker constituted of the GFP driven by a pathogen-inducible promoter, this new TRAP system enabled efficient isolation of translated RNA from pathogen-infected leaf cells.

PENGEMBANGAN ANTENA TELEX HYGAIN 2 METER BAND MENJADI 3 x 5/8 λ DI FREKUENSI 144-147 MHz UNTUK KOMUNIKKASI AMATIR MENGGUNAKAN SISTEM PEMBALIK PHASE

Penulisan ini membahas pengembangan antenna telex hy-gain V2R 2x5/8λ 2m band pada frequensi 144-146 Mhz menjadi 3x5/8λ, dengan metode menambahkan trap system pembalik pahsa dan penambahan elemen 5/8λ. Hal ini bertujuan agar antenna ini mendapatkan penguatan penerimaan Gain dan nilai return loss yang dihasilkan lebih baik. Dari table 2 dan tiga kali hasil pengukuran dan pengujian antenna telex Hy-gain V2R 2x5/8λ pada resonansi frequensi 144Mhz daya rata-rata yang diterima -35dBm, sehingga FUSB = 144.1 MHz dengan daya -38 dBm dan FLSB = 143.2 MHz dengan daya -38 dBm maka didapatlah bandwidth sebesar 200Khz. Sedangkan antena Hy-gain V2R yang dikembangkan dengan penggunaan trap sistem pembalik phasa menjadi 3 x 5/8 λ dari hasil tiga kali pengukuran pada tabel 3, pada resonansi frequensi 144Mhz mendapatkan daya sebesar -34dBm sehingga FUSB = 144.4 MHz dengan daya -37 dBm dan FLSB = 143.3 MHz dengan daya -37 dBm maka hasil pengembangan antena ini mendapatkan bandwidth 1.1khz. Hasil pengukuran dari pengembangan antena yang menggunakan sistem trap pembalik pahsa menghasikan Gain antenna sebesar 1dB dan retruns loss sebesar -4,15 dB, karena nilai return loss yang baik tidak lebih dari -9,54 dB.

Timor Deer (Cervus Timorensis) Arrest System Marind People with Tidal Local Wisdom

Deer are wildlife that has economic value because it can produce meat, leather, and velvet (young horn). Deer populations in nature have decreased due to uncontrolled wild poaching and damage to habitat. Indonesia has five types of deer, i.e., Cervus unicolor, Cervus timorensis, Axis kuhlii, Muntiacus Muntjak, and Axis axis (deer total). However, deer in Indonesia that have the opportunity to be empowered, there are several types, namely Cervus timorensis, Cervus unicolor and Axis axis, and in Merauke, especially in the had tribe0 has its distinctive characteristic in the process of catching deer. The purpose of the study was to know the pattern of deer arrest by the Malind tribe. The method used in this research is to conduct surveys and Wawan ways as well as observation of the field, the data in the analysis in a descriptive approach to conclude the investigation. The results showed that the pattern of deer caught in the Marind tribe traditionally done with snares, bamboo rinsing, as well as patterns carried out covering the forest burning Sawvana in the dry season of the cattle, besieged fire and dwelling on the land, and the season The community rained to the deck area (dense forest) that became the deer's dwelling and avoided the puddle of the sea, and the community captured it quickly. The chance of this research is the process of catching deer, in general, is done by the had in 2 ways that in the summer by burning and drought with the trap system.