Difference between revisions of "Part:BBa K3570006"

Revision as of 13:27, 25 October 2020

DPP1 upstream homologous sequence

Usage

DPP1 upstream homology arm part shall be used together with DPP1 downstream homology arm part (BBa_K3570007) to target a functional yeast integration locus. When DPP1 up put to 5' of the biobrick together with DPP1 downstream to the 3', the biobrick can be integrated into the S. cerevisiae's genome. It will do homologous recombination within the Diacylglycerol pyrophosphate phosphatase 1 (DPP1) gene.

This sequence was identified from a personal communication with Dr. Gilles Truan.

Experiments

Cloning of tHMG1 and CrtE production

Summary and cloning strategy:

The cloning strategy was to clone the blocks into two vectors before bringing them together in a unique plasmid. The blocks B14, B15 and B16 would be cloned in a pUC19 using InFusion method to form pUC19-B14B15B16. The other blocks B17, B18 and B19 would be cloned in another pUC19 using InFusion method to form pUC19-B17B18B19. pUC19-B17B18B19 would be used as a template vector to insert the sequence of B14B15B16 from pUC19-B14B15B16.

File:T--Toulouse INSA-UPS--2020 fig16.png

Figure 16: Cloning strategy

Results and discussion:

Construction of pUC19-B14B15B16:

The gblocks B14, B15 and B16 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (Figure 17).

File:T--Toulouse INSA-UPS--2020 fig18.png

Figure 17: PCR verification of the digested pUC19 and the three gblocks B14, B15 and B16 The expected strands are at 2.6kb, 0.4kb, 1.8kb and 1.0kb respectively

pUC19 was digested by SbfI - BamHI and prepared to receive the PCR products B14, B15 and B16 by InFusion. After transformation of Stellar cells, selection on ampicillin, and minipreps of 8 clones, we checked the restriction profiles of the constructions. The results were then verified by digestion with the enzyme SacI (Figure 18).

      File:T--Toulouse INSA-UPS--2020 fig18Acloning.png

      File:T--Toulouse INSA-UPS--2020 fig18Bcloning.png

Figure 18: Infusion verification: the expected sizes were 4.8kb and 1.2kb

We had six clones that had the expected profile.Since the sequence was valid, we had successfully obtained the first plasmid of our tHmg1-CrtE construction.
Built of the pUC19-B17B18B19

The gblocks B17, B18 and B19 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (Figure 19).

      File:T--Toulouse INSA-UPS--2020 fig19.jpeg
 

Figure 19: PCR verification of the digested pUC19 and gblocks B17, B18, B19

We digested the pUC19 vector by BamHI and EcoRI was done and purified the digested vector on gel. We proceeded to the InFusion reaction, transformation of Stellar cells, selection on ampicillin, and minipreps from 6 clones. The plasmids were assessed by restriction profiling with the enzymes BamHI and EcoRI.

File:T--Toulouse INSA-UPS--2020 fig20.png

Figure 20: InFusion verification: the expected sizes were 4.8kb and 2.6kb

Only one clone had the expected profile (figure X). We sent it to be sequenced by Eurofins and it was fortunately valid. We also had successfully obtained the second plasmid of our tHmg1-CrtE construction.

Built of tHmg1-CrtE:

The next step was to combine both plasmids by subcloning the fragment B14B15B16 into plasmid pUC19-B17B18B19.

To do this, we first extracted the DNA with the QIAGEN Plasmid Plus Midi Kit. Then, we digested both plasmids with SbfI and BamHI and purified with the Monarch Genomic DNA Purification Kit by NEB. The fragments were ligated together with T4 DNA ligase by NEB followed by a transformation into Stellar cells (ampicillin selection). Over the eight assessed colonies, two colonies presented the expected restriction profile when digested with SbfI and EcoRI (Figure 21).

File:T--Toulouse INSA-UPS--2020 fig21.png

Figure 21: Ligation verification: the expected size is 6.6kb and 2.6kb.

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Since the construction was successful, we proceeded to the next step. We followed the protocol given by one of our advisors, Anthony Henras, in order to get competent yeast cells for the transformation. The plasmid was digested with enzymes SbfI and EcoRI and purified to transform the yeast Saccharomyces cerevisiae. The yeast was then grown on YNB HIS- for 3 days. At the third try, we were able to observe around 20 colonies in our yeast transformation, about the same on the positive control and none on the negative control plate.

To verify our colonies we performed a genomic PCR using the TaKaRa PCR Amplification Kit, so we randomly chose eight clones from our transformation and one from the positive control plate (Figure 22).

File:T--Toulouse INSA-UPS--2020 fig22.jpeg

Figure 22: Transformation verification: the expected size is 1.2kb.

All clones have the expected size (1.2kb), and the control where we inserted pRS313 does not show any band. We have successfully integrated tHmg1 and CrtE into the yeast!

References

• S. cerevisiae genome, chromosome IV, DPP1 gene. GenBank: CP046084.1

Sequence and Features