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 GAPDH promoter (pGAP) is considered to be a strong constitutive promoter which is used by RNA polymerase II. U6 promoter(pU6) is used to drive the expression of homing guide RNA（hgRNA） in lineage tracing for eukaryotic systems.  We put pGAP in the upstream of pU6. The system can read the information of snRNAs out of the transcriptomic information by polyA tail.

Biology and Usage

pGAP is considered to be a strong constitutive promoter which is promoted by RNA polymerase II. RNA polymerase III uniquely transcribes small non-coding RNAs, including 5S rRNA , tRNAs, and other essential RNAs such as the U6 snRNA[1]. pU6 is used to drive the expression of sgRNA in lineage tracing for eukaryotic systems.  In our project, we use pU6 to transcribe small non-coding RNAs, hgRNA in CRISPR-Cas genome-editing system[2]. We use pGAP to transcribe the hgRNA and add a polyA tail. Therefore the hgRNA can not only work with CRIAPR/Cas9 system, but also work as barcodes. It enables us to read the lineage information out of transcriptomic information. It is known that RNA polymerase III transcription product does not have polyA and cannot be captured by OligodT when reading transcriptomic information. Therefore, when you need to confirm the information of Pol III transcription products together with transcriptomic information, you can use this double promoter system. Use pGAP to drive the transcription of RNA polymerase III genes and add polyA tail as well.

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 systems. The test is divided into two steps. First step: to test whether pGAP will affect the production and the function of gRNA. We put gRNA targeting ADE2 gene downstream of pU6 in both the experimental group and control group. Put pGAP upstream of U6 promoter only in the experimental group. Results showed that both of the two groups turn red, thus pGAP won’t affect the production and the function of gRNA. Second step: to test that whether gRNA can be reverse transcribed using oligodT as the primer. For both the experimental group and the control group, we extracted total mRNA of these purified red colonies by TRIzol. Then the mRNA was reverse transcribed using oligodT as the primer. To test whether gRNA can be transcribed, we performed PCR on reverse transcription products by two specific primers. Agarose gel electrophoresis were performed on the PCR product. There came out a correct band. Then we sequenced the products and get the anticipated results.

Experimental approach

1.Construct recombinant plasmid. Get pGAP from the genome of Cryptococcus neoformans. Insert it upstream of pU6 on PRH003 plasmid. 2.Transform the product (2.5μL) into DH5α competent cells（50μL）, grow cells on each agar plate (containing Ampicillin). Incubate plates at 37°C overnight. Monoclones are selected by colony PCR. Expanding culture colonies at 37℃ 200rpm, then extracting plasmids and sequencing. 3.Use Kpn1 enzyme to linearise the plasmids and transform them into Cryptococcus neoformans by electroporation. 4.The C. neoformans is spread on YNBA selection medium, and the transformants grow after being cultured in an incubator kept at 30℃ for 4 days. Then the culture is transferred to a refrigerator at 4℃. 5.Red colonies are selected and inoculated into YPD medium, then place in an incubator kept at 30℃ for 4 days. Finally it is kept at 4℃ refrigerator. 6.For both the experimental group and the control group, we first extract the total mRNA of these red colonies by TRIzol. Then the mRNA is reverse transcribed using oligodT as the primer. 7.To test whether gRNA can be transcribed, we perform PCR on the reverse transcription products by two specfic primers. Then sequencing the PCR product to further prove the success of our design.

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.