Team:ZJUT-China/Parts

Parts

All parts designed by ZJUT-China  revolved around the Cell-Free system. We designed a total of 19 basic parts and 23 composite parts for the detection of RNA biomarkers. You can get more information by accessing basic parts, composite parts and parts collection.

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Best New Basic Part: BBa_K3885124

Best New Composite Part: BBa_K3885311

Basic Parts

A total of 19 basic parts were built for the Cell-Free system myTXTL, including 4 promoters, 3 ribosomal binding sites and 12 expressed genes. We selected σ28 factor and P28 promoter (BBa_K3885101) to complex different ribosomal binding sites UTR1, UTR2 and UTR3 (BBa_K3885111, BBa_K3885112 and BBa_K3885113) to better express the downstream genes. The corresponding gRNA (BBa_K3885172) and igRNA(BBa_K3885171) were designed to detect RNA biomarkers.

The best part is σ28 (BBa_K3885124). σ28 is the most competitive of the σ factors. Another important feature is σ28 has strong specificity with its promoter P28. The leak through the promoter P28 is almost systematically below the detection limit. So the σ28 unit is the most efficient and the most specific sigma factor unit tested in this work.

You can click the part number for more detail in the following table.

Composite Parts

A total of 23 composite parts were established, including 15 parts with characterized function. We used P70a-UTR1-deGFP (BBa_K3885311) to characterize the intensity of complexed expression of P70a and UTR1, then used P70-σ28 (BBa_K3885202), P28-tetO-deGFP-ssrA (BBa_K3885352), P28-tetR-ssrA (BBa_K3885212) to establish our gene line to characterize whether our biomarkers have been detected.

The best composite part is P70a-UTR1-deGFP, P70a is an E. coli cytoplasmic extract, absent any remaining living E. coli cells, provides the Transcription-Translation (TX-TL) molecular machineries. When P70a is associated with UTR1, deGFP synthesis can reach 2 mg /mL (80 μM) in batch mode reaction, comparable to using the T7 promoter.

You can click the part number for detail in the following table.

Parts Collection

Our best collection of parts revolves around the Cell-Free system. By constructing plasmids suitable for expression in the Cell-Free system, we can more simply and efficiently characterize the content of RNA biomarkers in our samples, so as to quickly characterize the recovery response of the disease. The corresponding gRNA (BBa_K3885172) and igRNA (BBa_K3885171) were designed to detect RNA biomarkers in vitro.

We also had 21 composite parts expressed in Cell-Free system that were used to construct our gene lines. We used P70a-σ28 (BBa_K3885202), P28-tetO-deGFP-ssrA (BBa_K3885352), P28-tetR-ssrA (BBa_K3885212) to establish our gene line to characterize whether our biomarkers have been detected.

You can click the part number for detail in the following table.