Difference between revisions of "Part:BBa K3506095"

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Constitutive double promoter module

Constitutive double promoter system is composed of GAPDH promoter (BBa_K3506099) and U6 promoter (BBa_K3506021).

It is generally accepted that GAPDH promoter is a strong constitutive promoter which is recognized by RNA polymerase II.

U6 promoter is used to initiate the expression of homing guide RNA (hgRNA) in lineage tracing for eukaryotic systems.  We put GAPDH promoter in the upstream of U6 promoter. The system can read the information of hgRNA out of the transcriptomic information by polyA tail.

Biology and Usage

GAPDH promoter is considered to be a strong constitutive promoter. It is recognized by RNA polymerase II, which can add the polyA tail at 5' end of downstream genes.

U6 promoter is used to drive the transcription of small non-coding RNAs [1] and it is recognized by RNA polymerase III, which can not add the polyA tail at 5' end of downstream genes. 

In our project, we use U6 promoter to transcribe functional hgRNA without polyA tail [2]. We use GAPDH promoter to transcribe the DNA of U6 promoter and hgRNA and also add a polyA tail. Therefore, hgRNA can not only work with the CRISPR/Cas9 system but also be reverse transcribed by oligo dT. It enables us to obtain the lineage information together with transcriptomic information by single cell RNA sequencing.

Sequence and Feature

Design and Properties

We use U6 promoter (BBa_K3506021) to transcribe hgRNA, and GAPDH promoter (BBa_K3506099) is used to transcribe the DNA of U6 promoter and hgRNA.

We tested the constitutive double promoter module. This test is divided into two steps.

First step: to test whether GAPDH promoter will affect the production and the function of hgRNA. We put hgRNA targeting ADE2 gene downstream of U6 promoter in both the experimental group and the control group. A loss-of-function mutation in ADE2 can result in an adenine auxotroph that forms pink colonies on YNBA paltes containing a low level of adenine, thus enabling a visual evaluation of the action of CRISPR/Cas9. Put GAPDH promoter upstream of U6 promoter only in the experimental group. Results showed that both of the two groups turned red, thus GAPDH promoter won’t affect the production and the function of hgRNA.

Second step: to test that whether hgRNA can be reverse transcribed when using oligo dT as the primer. For both the experimental group and the control group, we extracted total mRNA of these pink colonies by TRIzol. Then the mRNA was reverse transcribed using oligo dT as the primer. To test whether hgRNA can be transcribed, we performed PCR on reverse transcription products by two specific primers. Agarose gel electrophoresis was performed on the PCR products. There came out a correct band (208bp). Then we sequenced the products and got the anticipated results.

Figure 2. Gel electrophoresis results of control group(pU6-gDNA) and experimental group(pGAP-pU6-gDNA). Lane 1: Marker; Lane 2 and Lane 3: RT-PCR product of control group(pU6-gDNA); Lane 4 and Lane 5: RT-PCR product of experimental group(pGAP-pU6-gDNA) (208 bp).

Experimental approach

1. Construct recombinant plasmid. Get GAPDH promoter from the genome of Cryptococcus neoformans. Insert it upstream of U6 promoter on PRH003 plasmid.

2. Transform the product (2.5μL) into DH5α competent cells（50μL）, grow cells on LB-amphenicol medium. Incubate 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 Cryptococcus 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. Pink colonies are selected and inoculated into YPD medium, then placed 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 pink colonies by TRIzol. Then the mRNA is reverse transcribed using oligo dT as the primer.

7. To test whether hgRNA can be reverse transcribed, we perform PCR on the reverse transcription products by two specfic primers. Then sequencing the PCR products to further prove the success of our design.

References

[1]Duttke S. H. (2014). RNA polymerase III accurately initiates transcription from RNA polymerase II promoters in vitro. The Journal of biological chemistry, 289(29), 20396–20404.

[2]Gao, Z., Herrera-Carrillo, E., & Berkhout, B. (2018). RNA polymerase II activity of type 3 pol III promoters. Molecular therapy. Nucleic acids, 12, 135–145.