Difference between revisions of "Part:BBa K3570008"
 
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<h2>Usage</h2>
 
<h2>Usage</h2>
 
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<p style="text-indent: 40px">
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HIS3 gene, found in the <i>Saccharomyces cerevisiae</i> 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.</p>
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HIS3 gene, found in the <i>Saccharomyces cerevisiae</i> yeast, encodes a protein called Imidazoleglycerol-phosphate dehydratase which catalyzes the sixth step in histidine biosynthesis(1). It is analogous to hisB in <em>Escherichia coli</em>.</p>
 
<p style="text-indent: 40px">
 
<p style="text-indent: 40px">
 
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]. </p>
 
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]. </p>
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<h2>Experiments</h2>
 
<h2>Experiments</h2>
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<p> We used this part to verify the integration of the tHMG1 and CrtE genes (part [https://parts.igem.org/Part:BBa_K3570000 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.</p>
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<p> We used this part to selection transformed colonies with the part [https://parts.igem.org/Part:BBa_K3570000 BBa_K3570000] (coding for tHMG1 and CrtE). Below is our yeast transformation protocol and our results which show that the BBa_K3570008 works.</p><br>
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<p><strong>Cloning of tHMG1 and CrtE</strong></p>
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<li>Summary and cloning strategy:</li>
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<p>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.</p>
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[[File:T--Toulouse_INSA-UPS--2020_CB-F1.png|500px|thumb|center|Figure 1: Cloning strategy]]
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<p> <strong>A. Protocols </strong></p><br>
  
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<li>Results and discussion:</li>
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<li><strong>Preparation of yeast competent cells</strong></li>
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<p>Construction of pUC19-B14B15B16:</p>
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<p>Fresh yeast were grown in 25 ml of YPD medium overnight. This preculture was diluted to low OD<sub>600nm</sub> (e.g. 0.05) in 50 ml of fresh YPD medium. The biomass concentration was measured every two hours until it reaches an OD<sub>600nm</sub> of around 0.8. The 50 ml of culture was transfered in a 50 ml falcon-tube and was centrifuged 5 minutes at 3000 rpm at room temperature. The supernatant was removed and 25 ml of LiAc/TE was added. The tube had to be thoroughly inverting 10 times. The tube was centrifuged 5 minutes at 3000 rpm at room temperature. The supernatant was removed and 400 µl of LiAc/TE was added. The tube had to be thoroughly inverting 10 times. Yeast competent cells should be used on the same day that they have been prepared.</p><br>
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<p>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).</p>
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[[File:T--Toulouse_INSA-UPS--2020_CB-F2.png|500px|thumb|center|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]]
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<li><strong>Yeast transformation</strong></li>
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<p>A mix in 1.5 ml microcentrifuge tube was prepared with 2 µl of transforming DNA (BBa_K3570000), 40 µl of competente yeast cells , 25 µg of carrier DNA (SS-DNA) and 168 µl of 50% PEG in 100 mM LiAc/TE.
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<p>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 <em>Sac</em>I (Figure 3).</p>
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Positive control was the same mixture but the transforming DNA was replaced by 1 µL of pR313 (a plasmid which have the HIS3 marker). The negative control was the same mixture but had no transforming DNA.
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After vortexing, the solution was incubated 45 minutes at 30 °C. Then 13 µl of DMS0 was added and the solution was vortexed again. It was centrifuged at 10,000 rpm for 1 minute. The supernatant was removed and the pellet was resuspended in 80 µl of NaCl. The solution was seeded on petri dishe of YNB with all amino acids expect histidine. The petri dishe was incubated at 30 °C for three days.</p><br>
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      [[File:T--Toulouse_INSA-UPS--2020_CB-F2.png|500px|thumb|center|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]]
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      [[File:T--Toulouse_INSA-UPS--2020_CB-F3.png|500px|thumb|center|Figure 3: Infusion verification: the expected sizes were 4.8kb and 1.2kb]]
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<p>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.<br>
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Built of the pUC19-B17B18B19</p>
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<p>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).</p>
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      [[File:T--Toulouse_INSA-UPS--2020_CB-F4.png|500px|thumb|center|Figure 4: PCR verification of the digested pUC19 and gblocks B17, B18, B19]]
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<p>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 <em>BamH</em>I and <em>EcoR</em>I.</p>
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[[File:T--Toulouse_INSA-UPS--2020_CB-F5.png|500px|thumb|center|Figure 5: InFusion verification: the expected sizes were 4.8kb and 2.6kb]]
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<p>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.</p>
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<p>Built of tHmg1-CrtE:</p>
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<p>The next step was to combine both plasmids by subcloning the fragment B14B15B16 into plasmid pUC19-B17B18B19.</p>
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<p>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 <em>Sbf</em>I and <em>EcoR</em>I (Figure 6).</p>
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[[File:T--Toulouse_INSA-UPS--2020_CB-F6.png|500px|thumb|center|Figure 6: Ligation verification: the expected size is 6.6kb and 2.6kb.]]
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<li>Yeast transformation:</li>
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<p>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 <em>Sbf</em>I and <em>EcoR</em>I 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.</p>
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<p>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).</p>
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[[File:T--Toulouse_INSA-UPS--2020_CB-F7.png|500px|thumb|center|Figure 7: Transformation verification: the expected size is 1.2kb.]]
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<p>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!</p>
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<strong>B. Results and discussion</strong><br>
  
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<p>We integrated our insert using the DPP1 integration locus. The strain BY4741 <em>Saccharomyces cerevisiae</em> was transformed with the insert and was grown on YNB with all amino acids expect histidine for 3 days. 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. This result prove that the HIS3 selectable marker works. </p>
  
 
<h2>References</h2>
 
<h2>References</h2>

Latest revision as of 18:08, 27 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 selection transformed colonies with the part BBa_K3570000 (coding for tHMG1 and CrtE). Below is our yeast transformation protocol and our results which show that the BBa_K3570008 works.


A. Protocols


  • Preparation of yeast competent cells

    • Fresh yeast were grown in 25 ml of YPD medium overnight. This preculture was diluted to low OD600nm (e.g. 0.05) in 50 ml of fresh YPD medium. The biomass concentration was measured every two hours until it reaches an OD600nm of around 0.8. The 50 ml of culture was transfered in a 50 ml falcon-tube and was centrifuged 5 minutes at 3000 rpm at room temperature. The supernatant was removed and 25 ml of LiAc/TE was added. The tube had to be thoroughly inverting 10 times. The tube was centrifuged 5 minutes at 3000 rpm at room temperature. The supernatant was removed and 400 µl of LiAc/TE was added. The tube had to be thoroughly inverting 10 times. Yeast competent cells should be used on the same day that they have been prepared.


  • Yeast transformation

    • A mix in 1.5 ml microcentrifuge tube was prepared with 2 µl of transforming DNA (BBa_K3570000), 40 µl of competente yeast cells , 25 µg of carrier DNA (SS-DNA) and 168 µl of 50% PEG in 100 mM LiAc/TE. Positive control was the same mixture but the transforming DNA was replaced by 1 µL of pR313 (a plasmid which have the HIS3 marker). The negative control was the same mixture but had no transforming DNA. After vortexing, the solution was incubated 45 minutes at 30 °C. Then 13 µl of DMS0 was added and the solution was vortexed again. It was centrifuged at 10,000 rpm for 1 minute. The supernatant was removed and the pellet was resuspended in 80 µl of NaCl. The solution was seeded on petri dishe of YNB with all amino acids expect histidine. The petri dishe was incubated at 30 °C for three days.


    B. Results and discussion

    We integrated our insert using the DPP1 integration locus. The strain BY4741 Saccharomyces cerevisiae was transformed with the insert and was grown on YNB with all amino acids expect histidine for 3 days. 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. This result prove that the HIS3 selectable marker works.

    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


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      INCOMPATIBLE WITH RFC[12]
      Illegal NheI site found at 1005
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal BglII site found at 896
      Illegal BglII site found at 956
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      COMPATIBLE WITH RFC[25]
    • 1000
      COMPATIBLE WITH RFC[1000]