Part:BBa_K3506095

Constitutive double promoter module

Constitutive double promoter system is composed of GAPDH promoter(BBa_K3506020) and CnU6 promoter(BBa_K3506021).

It is generally accepted that all poly(A) tail of eukaryotic cells is added by RNA polymerase II. GAPDH promoter (pGAP) is considered to be a strong constitutive promoter which is promoted by RNA polymerase II.

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]. Type 3 Pol III promoters have been widely used for synthesizing small functional RNAs, such as the shRNA in RNAi applications and gRNA in CRISPR-Cas genome-editing platforms[2]. 

We put GAPDH promoter in front of U6 promote(pU6).The system can not only transcribe small non-coding RNAs by U6 promoter, but also transcribe the U6 promoter and the genes downstream of U6 promoter by GAPDH promoter, which makes it possible to read the information of snRNAs out of out of transcriptomic information.

Biology and Usage

It is known that RNA polymerase III transcription product does not have polyA and cannot be captured by Oligo dT for information reading. Therefore, when you need to read Pol III transcription product information at the RNA level, you can use our dual promoter.Use GAPDH promoter to drive the transcription of RNA polymerase III promoter of the U6 small RNA gene and the genes downstream of U6 promoter.

Sequence and Feature

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 436
  

Properties

We tested the pU6 and pGAP system. The test is divided into two parts.

First part: to test whether pGAP will affect the production and function of gRNA. We put sgRNA which target ADE2 gene downstream of U6 promoter in both experimental group and control group. Put pGAPDH upstream of U6 promoter only in experimental group. Result shows that pGAP won’t affect the production and function of gRNA, because both of the two groups turn red.(图)

Second part: to test that whether gRNA can be reverse transcribed by oligodT. For both experimental group and control group, we extract the total mRNA of these red colonies by TRIzol. Then the mRNA was reverse transcribed by oligodT. To test whether gRNA can be transcribed, we performed PCR on reverse transcription products by two specfic primers. Agarose gel electrophoresis were performed on the PCR product. There came out a correct band(图）. Then we sequenced the products and get the right result.

Experimental approach

1.Construct recombinant plasmid. Get pGAP from the genome of Cryptococcus neoformans. Inserted it upstream of pU6 on PRH003 plasmid.

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.

【2】Gao Z, Herrera-Carrillo E, Berkhout B. RNA Polymerase II Activity of Type 3 Pol III Promoters. Mol Ther Nucleic Acids. 2018 Sep 7;12:135-145.