Difference between revisions of "Part:BBa K3570008"
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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 experiments 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 in the insertion of the tHMG1 and CrtE genes (part [https://parts.igem.org/Part:BBa_K3570000 BBa_K3570000]) in the yeast genome. Below is our yeast transformation protocol and our results which show that we have successfully integrated this part and that the BBa_K3570008.</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><br>
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<p>Construction of pUC19-B14B15B16:</p>
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<p>Fresh yeast were grown in 25ml of YPD medium overnight. This preculture was diluted to low OD<sub>600nm</sub> (e.g. 0.05) in 50ml 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 50ml of culture was transfered in a 50ml falcon-tube and was centrifuged 5 minutes at 3000rpm at room temperature. The supernatant was removed and 25ml of LiAc/TE was added. The tube had to be thoroughly inverting 10 times. The tube was centrifuged 5 minutes at 3000rpm at room temperature. The supernatant was removed and 400µl 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><br>
 +
<p>A mix in 1.5ml microcentrifuge tube was prepared with 2µl of transforming DNA (BBa_K3570000), 40µl of competente yeast cells , 25µg of carrier DNA (SS-DNA) and 168µl of 50% PEG in 100mM LiAc/TE.
 +
Positive control was the same mixture but the transforming DNA was replaced by 1uL 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 13µl 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 80µl 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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<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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<li><strong>Integration verification PCR</strong></li><br>
 +
<p>The integration of BBa_K3570000 using the DPP1 homology sequence (BBa_K3570006 and BBa_K3570007) was performed by a genomic PCR using the TaKaRa PCR amplification Kit. Our insert was to big to be amplified by PCR. We therefore decided to amplified a part of 1200kb in the genome. The following primers have been used. The primer 1 (forward) hybridizes on our selectable marker HIS3 . The primer 2 (reverse) hybridizes on the 1,039,876 pb of the chromosome IV of the strain BY4741 (upstream of the DPP1 gene).</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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<strong>B. Results and discussion</strong><br>
  
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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>Since the construction of the part BBa_K3570000 was successful, we proceeded to the next step: integration in the yeast genome. The plasmid was digested with enzymes SbfI and EcoRI and purified to transform the yeast Saccharomyces cerevisiae resulting in th strain BY4741 DPP::tHMG1-crtE. The yeast was then grown on YNB with all amino acids expect histidine 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. We randomly chose eight clones from our transformation and one from the positive control plate (see on the figure 1 below) for PCR verification integration.</p>
  
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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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[[File:T--Toulouse_INSA-UPS--2020_CB-F7.png|500px|thumb|center|Figure1: Verification of the integration of the part BBa_K3570000 in the yeast genome using DPP1 homology sequence (BBa_3570006 and BBa_3570007). The expected size is 1.2kb.]]
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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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−
 
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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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−
 
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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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−
 
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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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−
 
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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>
+
  
 +
<p>All clones have the expected size (1.2kb), and the control where we inserted pRS313 does not show any band.<br>
 +
The integration of BBa_K3570000 should results in an increase of the GGPP quantity. For further verification, we analyzed the quantity of GGPP in the strain BY4741 DPP::tHMG1-crtE and in the wild type.</p><br>
  
 +
These two verifications proved that we have successfully integrated tHmg1 and CrtE into the yeast using the selectable marker HIS3!</p>
  
 
<h2>References</h2>
 
<h2>References</h2>

Revision as of 10:44, 26 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 in the insertion of the tHMG1 and CrtE genes (part BBa_K3570000) in the yeast genome. Below is our yeast transformation protocol and our results which show that we have successfully integrated this part and that the BBa_K3570008.


A. Protocols


  • Preparation of yeast competent cells

    • Fresh yeast were grown in 25ml of YPD medium overnight. This preculture was diluted to low OD600nm (e.g. 0.05) in 50ml of fresh YPD medium. The biomass concentration was measured every two hours until it reaches an OD600nm of around 0.8. The 50ml of culture was transfered in a 50ml falcon-tube and was centrifuged 5 minutes at 3000rpm at room temperature. The supernatant was removed and 25ml of LiAc/TE was added. The tube had to be thoroughly inverting 10 times. The tube was centrifuged 5 minutes at 3000rpm at room temperature. The supernatant was removed and 400µl 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 1.5ml microcentrifuge tube was prepared with 2µl of transforming DNA (BBa_K3570000), 40µl of competente yeast cells , 25µg of carrier DNA (SS-DNA) and 168µl of 50% PEG in 100mM LiAc/TE. Positive control was the same mixture but the transforming DNA was replaced by 1uL 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 13µl 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 80µl 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.


  • Integration verification PCR

    • The integration of BBa_K3570000 using the DPP1 homology sequence (BBa_K3570006 and BBa_K3570007) was performed by a genomic PCR using the TaKaRa PCR amplification Kit. Our insert was to big to be amplified by PCR. We therefore decided to amplified a part of 1200kb in the genome. The following primers have been used. The primer 1 (forward) hybridizes on our selectable marker HIS3 . The primer 2 (reverse) hybridizes on the 1,039,876 pb of the chromosome IV of the strain BY4741 (upstream of the DPP1 gene).


    B. Results and discussion

    Since the construction of the part BBa_K3570000 was successful, we proceeded to the next step: integration in the yeast genome. The plasmid was digested with enzymes SbfI and EcoRI and purified to transform the yeast Saccharomyces cerevisiae resulting in th strain BY4741 DPP::tHMG1-crtE. The yeast was then grown on YNB with all amino acids expect histidine 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. We randomly chose eight clones from our transformation and one from the positive control plate (see on the figure 1 below) for PCR verification integration.

    Figure1: Verification of the integration of the part BBa_K3570000 in the yeast genome using DPP1 homology sequence (BBa_3570006 and BBa_3570007). 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.
    The integration of BBa_K3570000 should results in an increase of the GGPP quantity. For further verification, we analyzed the quantity of GGPP in the strain BY4741 DPP::tHMG1-crtE and in the wild type.


    These two verifications proved that we have successfully integrated tHmg1 and CrtE into the yeast using the selectable marker HIS3!</p>

    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]