Difference between revisions of "Part:BBa K3506022"

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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 drive 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.

U6 promoter is used to drive the transcription of small non-coding RNAs [1] and it is recognized by RNA polymerase III.

In our project, we use U6 promoter to transcribe hgRNA in CRISPR-Cas genome-editing system constitutively[2]. We use GAL7 promoter to transcribe the hgRNA at a specific time and add a polyA tail when induced. So the hgRNA can not only work with CRISPR/Cas system but also work as the barcode. It enables us to read the lineage information in hgRNA out of transcriptomic information.

It is known that the RNA polymerase III transcription product does not have polyA, nor can it be captured by Oligo dT for information reading. Therefore, you can use our double promoter system when you need to read the information of Pol III transcription product together with transcriptomic information at a specific time. This is very significant for knowing the functions and influences of this kind of RNA.

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.