Part:BBa_K3506022
Inducible double promoter system
GAL7 promoter can be induced by galactose in Cryptococcus neoformans. It is the first inducible promoter characterized in C. neoformans. RNA polymerase III promoter of the U6 small RNA gene is a common part for eukaryotic expression systems. Pol III uniquely transcribes small non-coding RNAs, including 5S rRNA, tRNAs, and other essential RNAs such as the U6 snRNA【1】. But it is known that RNA polymerase III transcription product does not have polyA and cannot be captured by Oligo dT for information reading.We designed this composite part to read the sequence information downstream of the U6 promoter in real time and at RNA level through the drive of GAL7 promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 402
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 742
Experimental approach
1.Construct recombinant plasmid. Get pGAL7 from the PYES2 plasmid. Inserted it upstream of pU6 on PRH003 plasmid. Ligate the fragments by in-fusion cloning. 2.Transform the product (2.5μL) into DH5α competent cells（50μL）, coat cells on each agar plate (containing Ampicillin). Incubate plates at 37°C overnight. Monoclones were selected for colony PCR. Expanding culture colonies at 37℃ 200rpm,extract plasmids and sequence. 3.Use Kpn1 enzyme to linearise the plasmid and transformed it into Cryptococcus neoformans by electroporation. 4.The C. neoformans was spreed on YNBA selection medium, and the transformants grew after being cultured in an 30℃ incubator for days. Then transferred them to a 4℃ refrigerator. 5.After that, red single colonies were observed. Red colonies were selected and inoculated into YPD medium, then placed it in 30℃ incubator for days, and placed it in 4℃ refrigerator again. 6.After that, more red single colonies were observed. There was no significant difference in color between the experimental group and the control group(transformed the linearized PRH003 plasmid without GAPDH promoter). These proved that pGAP won't influence the original function of pU6 and gRNA.
References
[1]Duttke, S. H C . RNA polymerase III accurately initiates transcription from RNA polymerase II promoters in vitro.[J]. Journal of Biological Chemistry, 2014, 289(29):20396.
| None |