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Inducible double promoter system

Inducible double promoter system is composed of GAL7 promoter(BBa_K3506424) and U6 promoter(BBa_K3506021). GAL7 promoter can be induced by galactose in Cryptococcus neoformans. It is the first inducible promoter characterized in Cryptococcus neoformans. U6 promoter is used to initiate the expression of homing guide RNA（hgRNA) in lineage tracing for eukaryotic systems. 

We put GAL7 promoter in the upstream of U6 promoter. The system can read the information of hgRNA out of transcriptomic information by polyA tail.

Biology and Usage

GAL7 promoter can be induced by galactose and it is recognized by RNA polymerase II, which can add the polyA tail at 5' end of downstream genes.

U6 promoter is used to initiate 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, U6 promoter is used to transcribe hgRNA constitutively [2], which can be combined with Cas9. GAL7 promoter is used to transcribe the DNA of U6 promoter and hgRNA at a specific time. So we can add the polyA tail to hgRNA when induced, which enables it to be captured by oligo dT in single cell RNA sequencing. hgRNA works as the barcode. Thus, we can obtain the lineage information together with transcriptomic information by single cell RNA sequencing.

You can use our double promoter module when you need to read the information of Pol III transcription products together with transcriptomic information at a specific time. This is very significant for knowing the functions and influences of this kind of RNAs.

Sequence and Features

Design and Properties

We use U6 promoter (BBa_K3506021) to transcribe hgRNA, and GAL7 promoter (BBa_K3506424) is used to transcribe the DNA of U6 promoter and hgRNA when induced by galactose.

we tested the pU6 and pGAL7 system. The test is divided into two parts.

First part: to test whether pGAL7 will affect the production and function of hgRNA. We put sgRNA which target ADE2 gene downstream of U6 promoter in both experimental group and control group. Put pGAL7 upstream of U6 promoter only in experimental group. Result shows that pGAL7 won’t affect the production and function of gRNA, because both of the two groups turn red.(Figure 1.)

Second part: to test that whether gRNA can be reverse transcribed by oligo dT. 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 oligo dT. 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(Figure 2.). Then we sequenced the products to prove the success of our engineer further.

Fig. 1 A. control group(pU6-gDNA); B. experimental group(pGAL7-pU6-gDNA); C. and D. 4500FOA (the recipient strain)

Fig. 2 Gel electrophoresis results of control group(pU6-gDNA) and experimental group(pGAL7-pU6-gRNA). 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(pGAL7-pU6-gDNA) (208 bp).

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.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.For both experimental group and control group, we selected red colonies, induced by galactose for 30mins, extract the total mRNA by TRIzol. Then the mRNA was reverse transcribed by oligo dT.

7.To test whether gRNA can be transcribed, we performed PCR on reverse transcription products by two specfic primers. Then sequence the PCR product to proved the success of our design further.

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.