Difference between revisions of "Part:BBa K3570008"

Revision as of 14:12, 25 October 2020

HIS3 selection marker

Usage

HIS3 gene, found in the Saccharomyces cerevisiae yeast, encodes a protein called Imidazoleglycerol-phosphate dehydratase which catalyzes the sixth step in histidine biosynthesis(1). It is analogous to hisB in Escherichia coli.

HIS3 gene serves as a commonly used yeast selectable marker. When HIS3 gene is inserted into an integrative or replicative plasmid, HIS3 allows to counter-select the cells that acquired the prototroph character for histidine so that they can grow without histidine addition in the medium. Those cells should not have the functional HIS3 gene in its genome[1].

The sequence contains HIS3 specific promoter, HIS3 coding sequence, and HIS3 terminator. This sequence was taken from RS313 plasmid [3].

Experiments

We used this part to verify the integration of the tHMG1 and CrtE genes (part BBa_K3570000) in the yeast genome. Below is our cloning strategy and our experiences which show that we have successfully integrated this part and that the BBa_K3570008 part works.

Cloning of tHMG1 and CrtE

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.

Figure 1: Cloning strategy

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 2).

Figure 2: 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 3).

Figure 2: 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

Figure 3: 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 4).

Figure 4: 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.

Figure 5: InFusion verification: the expected sizes were 4.8kb and 2.6kb

Only one clone had the expected profile (figure 5). 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 6).

Figure 6: Ligation verification: the expected size is 6.6kb and 2.6kb.

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 7).

Figure 7: 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

• [1]- Old, R. W., & Primrose, S. B. (1981). Principles of gene manipulation: an introduction to genetic engineering (Vol. 2). Univ of California Press.
• [2]- GenBank: U03439.1
• [3]- RS313 plasmid
• [4]- SGD:S000005728

Sequence and Features