Difference between revisions of "Team:Paris Bettencourt/Notebook/Phytase"
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<div style="clear:both"></div> | <div style="clear:both"></div> | ||
− | Third step, we transformed yeast that is already integrated the resitance instead of PHO85 gene.<br> | + | Third step, we transformed yeast that is already integrated the resitance instead of PHO85 gene. We introduce the CRE and RFP gene instead of PHO 80 gene<br> |
The protocole is:<a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/Heat_shock_transformation_for_yeast"> transformation of yeast</a><br> | The protocole is:<a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/Heat_shock_transformation_for_yeast"> transformation of yeast</a><br> | ||
+ | Fourth step, we cultived the transforming yeast in plate with geneticin during all night. | ||
<h2> Annalyse the result Acid phytique tests</h2> | <h2> Annalyse the result Acid phytique tests</h2> |
Revision as of 16:02, 9 September 2015
Ferment It Yourself
iGEM Paris-Bettencourt 2O15
- Background
- Design
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Notebook
Vitamin A Vitamin B2 Vitamin B12 Phytase Riboswitch Differentiation on E. coli Differentiation on S. cerevisiae Manufacturing Idli and Micro-organisms August 8th
Design primers
Gene PHO85
5’Primer of Kanamycin resistance gene with tails using to transformation with the PHO85 gene of the yeast.
5’-TATCATTATATATACATGGCTACGGTTTTTCGCTGACGGGCTGCGATAATCATTTGCA TCCATACATTTTGATGGC -3’
3’Primer of Kanamycin resistance gene with tails using to transformation with the PHO85 gene of the yeast.
3’-AAGGGATATATAGCGCGGCAAACTGGGCAAACTTGAGCAATACCACAGCAGTATAG CGACCAGCATTC-5’
- Homology tail on gene PHO85
- Kanamycin resistance binding
Gene PHO80
5’Primer of Kanamycin resistance gene with tails using to transformation with the PHO80 gene of the yeast.
5’-ATCATAAGACGAGGATATCCTTTGGAGACTCATAGAAATCATAATCATTTGCATCCAT ACATTTTGATGGC-3’
3’Primer of Kanamycin resistance gene with tails using to transformation with the PHO80 gene of the yeast.
3’-CTCAATCATGATTGCTTTCATAATACCCCACGAAAAATCACAGCAGTATAGCGACCA GCATTC-5’
- Homology tail on gene PHO80
- Kanamycin resistance binding
Gene FRT + PHO85
5’Primer of Kanamycin resistance gene with tails using to transformation with the PHO85 gene of the yeast, including FRT sequence to delete both of PHO80 and PHO85.
5’-TATCATTATATATACATGGCTACGGTTTTTCGCTGACGGGCTGCGGAAGTTCCTATTC TCTAGAAAGTATAGGAACTTCATAATCATTTGCATCCATACATTTTGATGGC-3’
3’Primer of Kanamycin resistance gene with tails using to transformation with the PHO85 gene of the yeast, including FRT sequence to delete both of PHO80 and PHO85.
3’-AAGGGATATATAGCGCGGCAAACTGGGCAAACTTGAGCAATACCACTTCAAGGATAT GAAAGATCTCTTATCCTTGAAGCAGCAGTATAGCGACCAGCATTC-5’
- Homology tail on gene PHO85
- FRT sequence
- Kanamycin resistance binding
August 12nd
Culture
Inoculate 100µL of Saccharomyces cerevisiae SK1 on YPD medium overnight (at 30°C).
This yeast will be transformed.
PCR
3 PCR were realized on HO-Poly-KanMX4-HO plasmid to create a Kanamycin resistance marker, thanks to 3 pairs of primers wich have tails we’ll be use to knock out genes PHO80, PHO85 and both in the yeast.
Protocol:
PHO80 PHO85 FRT+ PHO85 Master mix (µL) 50 50 50 H2O DNAse Free (µL) 45 45 45 Resistance plasmid (µL) 1 1 1 PHO80 5'Primer (µL) 2 PHO80 3'Primer (µL) 2 PHO85 5'Primer (µL) 2 PHO85 3'Primer (µL) 2 PHO85 + FRT 5'Primer (µL) 2 PHO85 + FRT 3'Primer (µL) 2 Figure 1 : PCR cycle
August 13th
PCR Purification
Protocol : PCR purification
Result of PCR (August 12th)
The PCR product are then revealed with a gel electrophoresis.
We expected bands around 1.300bp. The band corresponding to marker with FRT is bigger than the two others strips because these have just the Kanamycin resistance with tails, and no FRT sequences.
Figure 2 :Result of PCR
Pre-culture
Plated one colony of Saccharomyces cerevisiae SK1 in 5mL liquid YPD medium and let's grow overnight.
August 14th
Transformation of yeast
Protocol: Heat shock transformation for yeastAugust 17th
Result of plates:
There is a culture in plates.
The negative control is not well. The no change yeast grow in the YPD medium with the antibiotic.
We will repeat this control on an agar plate and not in a liquid medium.
We analyze anyway down results, the results of the new control will allow us to validate the result of our experiment or search which are our error and try again.
The positive control is well, yeast multiply on YPD medium plate without antibiotic. Yeasts are not dead, so the culture on other agar mediums are not contamination.
We see more colonies on the plates with yeast transforming PHO85 and FRT+PHO85.
We look only few colonies in the plates with yeast transforming PHO80.
The result is well, transformation works.
Figure 3 : Negative control
Figure 4 : Positive control and result of transformation
Design primers
We design primers to verifie our results on the plates.
Thanks to the colony PCR, we might determinate if the resistance is integrated into the yeast DNA.
Primer 5'-3' PHO80
ATCATAAGACGAGGATATCCTTTGGAG
Primer 3'-5' PHO80
CTCAATCATGATTGCTTTCATAATACCCC
Primer 5'-3' PHO85
TATCATTATATATACATGGCTACGGTTTTTCG
Primer 3'-5' PHO85
AAGGGATATATAGCGCGGCAAACTG
Primer 5'-3' FRT+PHO85
TATCATTATATATACATGGCTACGGTTTTTCG
Primer 3'-5' FRT+PHO85
AAGGGATATATAGCGCGGCAAACTG
August 18th
Verification of the new negative control
The verification of the negative control is good, any colony is watching. We can continue our experiments, it will be validated.
Figure 5 : Result of the new negative control
FRT problems
The transformation with the FRT may be run well, but the plasmid with the gene coding for flippase works only for E. coli. We can't use this plasmid because it will be rejected by the yeast.
Other transformation with CreLox system is possible.
CreLox is a gene which have the same fonction than the FRT, it not cut by the flippase but by the Cre recombinase.
We design two primers for the new transformation with CreLox system.
Primer 5'-3' CreLox + PHO85
5’-TATCATTATATATACATGGCTACGGTTTTTCGCTGACGGGCTGCGATAACTTCGTATAGCATACATTATACGAAGTTATATAATCATTTGCATCCATACATTTTGATGGC-3’
Primer 3'-5' CreLox + PHO85
3’-AAGGGATATATAGCGCGGCAAACTGGGCAAACTTGAGCAATACCAATAACTTCGTATAGCATACATTATACGAAGTTATCAGCAGTATAGCGACCAGCATTC-5’
- Homology tail on gene PHO85
- CreLox sequence
- Kanamycin resistance binding
August 19th
Colony PCR
Protocol:
PHO80 PHO85 FRT+ PHO85 dreamTaq 2X (µL) 3 3 3 H2O DNAse Free (µL) 9 9 9 Colony 1 1 1 PHO80 5'Primer (µL) 0.5 PHO80 3'Primer (µL) 0.5 PHO85 5'Primer (µL) 0.5 0.5 PHO85 3'Primer (µL) 0.5 0.5 Figure 6 : Colony PCR cycle
August 20th
Result of colony PCR (August 19th)
We only see bands smaller than 500bp, but not the fragments we expected : this is probably contaminations. We start again the same PCR colony.
Figure 7 : Electrophoresis PCR colony
Colony PCR
Same to August 19th.Result of second colony PCR
Figure 8 : second electrophoresis colony PCR
The DNA Ladder is good but DNA of PCR did not migrate. The ADN is in the holes. We think that the yeasts walls being thicker simple thermic shock does not break it. We will be carry a lysis whith NaOH in yeast, to push out DNA, to the primer can be fixed on it.
August 24th
Yeast lysis with NaOH
Protocol: Yeast lysis with NaOH
After the lysis of yeast we realize the new PCR in normal condition, the same as August 12nd.
August 25th
Result of PCR (August 24th)
Figure 9 : Third electrophoresis colony PCR with NaOH lysis
The DNA ladder migrate, but there was any amplification of the both genes.
We tested two PCR mix : the first did not work. The positive control worked with the second PCR mix. It is composed of OH plasmid and the oligos using for PHO85 gene.August 26th
Colony PCR
To make the colony PCR, we need to lysis yeasts' wall. We realized the lysis with NaOH, but it did not work. So we realize a new lysis using the " DNeasy Blood and Tissue Kit" with the zymolyase enzyme on the non-transformed yeasts to verifie the melting temperature of primers (try between 55°C and 65°C) and if the amplification of the genes work.
Phytic acid dosage
We dose the phytic acid in the fermented rice with the kit " Phytic Acid (Total Phosphorus) Assay Kit ".August 27th
Result of PCR (August 26th)
Figure 10 : Electrophoresis colony PCR with temperature gradient on non-transformed yeasts lysed by the zymolyase
We made a PCR gradient on non-transformed yeast to know exactly wich temperature is better to a good fixation of the primers on the DNA.
The amplification failed, we supposed it is because our MasterMix did not work. We will try this PCR again.Phytic acid dosage
We dose the phytic acid in fermented rice.
Figure 12 : Acid phytic dosage on fermented rice
Result of second PCR
Figure 11 : Second electrophoresis colony PCR with temperature gradient (non-transformed yeast)
We watch bands for the gene PHO80, at the good size : 882bp. But the gene PHO85, there was no amplifiction, and the positive control is negative : we only see aband bigger than 10,000bp and it is not what we expected.
We try again this PCR to see if the no amplification of the gene PHO85 it is a manipulation error or not.August 28th
Phytic acid dosage in different strains
Figure 13 : Acid phytic dosage on fermented rice with some strains
Figure 14 : Results of acid phytic dosage on fermented rice
Results of PCR
Figure 15 : Third electrophoresis gradient PCR (non-transformed yeast)
We watch bands for the both of genes, but not at the same temperatures. Thanks to the gradient, we can suppose that oligos have not exactly the same melting temperature, and it may be the reason why the previous PCR failed.
The controle postive with the OH plasmid worked, the band matches with the PHO85 gene size (1020bp).
Figure 16 : Electrophoresis PCR transformed yeasts
the positive control is negative PCR does not therefore our work.
Transformation
We transform the yeast (...) with the resistance to geneticin and the CRE gene. We delete the PHO 85 genes and we replace it by the resistance gene.
Figure 16 :Principle of Cre recombinase
The CRE gene is the sequence that little be cut thanks Cre recombinase and reconbined the gene without the sequence between the CRE gene.
The protocole is: transformation of yeastAugust 30th
The transformation on yeats with CRE gene
Figure 16 : Plate YPD + geneticin plated with transforme yeast
We watching Any culture in the plate YPD + geneticin. The yeast is don't resistante to geneticin, the yeast has not introduced the resistance gene. Transformation does not work.
September 2nd
Transformation
We start again the transformation of august 28th, with the same protocol.Teste of acid phytic
We test the quantity of acid phytic in the 915,37 dilution 1/5, 915,37 dilution 1/50, 915,37 dilution 1/500, 915,55 dilution 1/5, 915,55 dilution 1/50, 915,55 dilution 1/500, 915,56 dilution 1/5, 915,56 dilution 1/50, 915,56 dilution 1/500, 915,57 dilution 1/50, 915,57 dilution 1/500, 915,58 dilution 1/5, 915,58 dilution 1/50, 915,58 dilution 1/500, Sook dall + HCl and Sook rice + HClSeptember 4th
After the transformation of september 2nd, we see the plate but any culture is observable. The tansformation are not function. We restart transformation, but we transforme with CRE and RFP gene. It is more interesting because we not forced to simply remove the resistance gene, we can make a double identification.It helps identify yeast that is already integrated in place of the resistance gene Pho85 (they cultivate on agar geneticin) and we also integrate the RFP gene in place of PHO80 gene (they will be red).September 8th
Transformtion with the CRE and RFP gene
We restarst transformation of septembre 2nd, with CRE and RFP gene.
First step, we realize the PCR of CRE and RFP gene withe the primer (creats by Amaury) and plasmid RFP. We amplified the gene, its promoter and its terminater.
The protocole is: PCR
Second step, we make PCR purification, and check the result with electrophorese
The protocole is: PCR purification
Figure 17 :electrophorese of CRE and RFP amplification
Third step, we transformed yeast that is already integrated the resitance instead of PHO85 gene. We introduce the CRE and RFP gene instead of PHO 80 genewe see one bande of the frist line, this PCR has work well. we can continu this experience and realize the transformation
The protocole is: transformation of yeast
Fourth step, we cultived the transforming yeast in plate with geneticin during all night.Annalyse the result Acid phytique tests