Yeast Lab: Cloning

Question 1

Goal

Answer 1

• To clone a higher plant gene by complementing a yeast mutant strain
• Attempting to clone a gene from the higher plant Arabidopsis thaliana
• Plant: Arabidopsis thaliana gene
  
  • homolog of the yeast MET3 gene
  • encodes ATP sulfurylase
    
    • catalyzes essential step in cysteine and methionine biosynthesis
• Yeast: met3 mutant
  
  • unable to synthesize methionine, thus requires it in the growth medium
• genetic complementation
  
  • when donor gene (plant gene) is expressed in recipient (yeast)
  • it can substitute the mutant recipient (yeast) gene
  • both donor and recipient must share the same pathway
  • recipient must be lacking the gene or have a mutation in the gene
  • introduced gene must be transcribed and RNA translated in expression vector
  • protein must be functional
  • frequently used method to obtain a desired clone from various organisms without knowing the sequence of the gene or protein in question

Question 2

Genetic Transformation

Answer 2

• Genetic material of a cell is altered by the introduction of exogenous DNA
  
  • can be from the same species or different organism
• Method
  
  • Heat shock
  • Electroporation
  • Microprojectile bombardment

Question 3

Electroporation

Answer 3

• more effective than heat shock
• A short electrical pulse
• disturbs the phospholipid bilayer of cell membrane resulting in temporary pores
• charged molecules (DNA) can enter via these pores
• Process
  
  • Pellet yeast cells
  • Wash in water to remove salts
    
    • does not work well with large qtys of salts
  • Resuspend in 1 M sorbitol as osmotic stabilizer
    
    • carbohydrate alcohol
  • Electroporate
  • Plate on selective medium - only transformants can grow

Question 4

Glucose and Galactose

Answer 4

• both carbohydrates
• glucose is preferred by yeast
• vary only in the orientation of OH group at one position
• Although they are similar in structure, galactose musts be converted to glucose to be used by cells.

Question 5

cDNA library of A. thaliana

Answer 5

• cDNA library of A thaliana was constructed to clone the homolog of the yeast MET3 gene
  
  • MET3 gene encodes ATP sulfurylase
  • catalyzes an essential step in cysteine and methionine biosynthesis
• complimentary DNA
• prepared from polyadenylated mRNA of expressed genes are obtained from A. thaliana (≈ 26,000 genes) to create cDNAs
• mRNA collected varies depending on the tissue the sample was taken from and what it was expressing at the time of collection
• One way to generate a cDNA
  
  • RNA is the template
  • Oligo (dT) primer is used to bind to the Poly(A) tail (‘3) of an RNA
  • Reverse transcriptase uses RNA as the template, and generates DNA
  • DNA is complimentary to the RNA
  • DNA polymerase I is then used to convert the new ssDNA to dsDNA
  • Sites are then added to the ends of the dsDNA
    
    • i.e. restriction enzyme sites
    • we added Xho1 linkers
    • Short dsDNA oligomers containing the same required restriction sites used to cut the plasmid
    • Aids with the insertion of the library into the vector
    • Allows cDNA to anneal to the plasmid
  • dsDNA is ligated in a cloning vector (plasmid)
    
    • we are using the Lambda YES vector
    • Each clone generally represents 1 mRNA
    • Abundance of a particular clone in the library reflects abundance of that mRNA in the sample
• Each plasmid containing 1 cDNA
• LIbraries can be transformed into bacteria, phage or yeast
• Transformants generally have 1 plasmid
• Expression of cDNA requires
  
  • full length (gene) cDNA
  • Promoter upstream of cDNA
  • for RNA polymerase binding

Question 6

Lambda YES vector

Answer 6

• designed to allow protein expression from a cDNA cloned downstream of the GAL1 promoter
• shuttle vector
  
  • Has selectable markers and can replicate in Escherichia coli or yeast
  • E. coli replication elements
    
    • Origin of replication (ori pBR322)
    • Ampicillin resistance for selection
  • S. cervisiae replication elements
    
    • yeast origin of replication (ARS - autonomously replicating sequence)
    • yeast centromere (CEN4) mitotic stability
• It contains Xhol restriction sites
  
  • cDNAs are cloned into the Xhol site, between the GAL1 promoter and HIS3 transcriptional terminator
  • cDNA library of A. thaliana is inserted here
• GAL1
  
  • regulated yeast promoter
  • active in yeast when galactose (carbon source) is present
  • inactive if glucose (carbon source) is present
• Ampicillin resistance gene
  
  • selectable marker
• yeast URA3 gene
  
  • allows for selection of transformants via a nutritional marker (common to do this)

Question 7

GAL1

Answer 7

• gene coding for galactokinase
• GAL1 promoter is upstream of the GAL1 gene and it’s induced by galactose
• When galactose enters a cell, it induces the GAL1 promoter to express the GAL1 gene resulting in the conversion of galactose to glucose-6-P, which can be utilized by the cell
• any gene inserted after the promoter will also be expressed

Question 8

A. thaliana cDNA was inserted into Lambda YES between GAL1 promoter and HIS3 transcriptional terminator.

Answer 8

Lambda Yes was selected because

• capable of expressing genes in both E. coli and S. cerevisiae
  
  • S. cerevisiae is used to maintain the plasmid (due to low copy numbers)
  • E. coli is used to replicate the plasmid (high copy number due to origin of replication)
• cDNA libraries inserted in Lambda YES can be used to isolate A. thaliana genes, by complementation of E. coli and yeast mutations
  
  • facilitates gene isolation from eukaryotes by bacterial and yeast complementation
• Has two selectable markers
  
  • antibiotic resistance (Ampicillin resistance) and URA3 gene marker
  • Both used to distinguish between transformants and non-transformants. For example:
    
    • Transformed cells w/the selectable marker can grow in the selectable medium but non-transformants can’t
    • Detection of recombinants w/selectable markers can be verified by gene expression of URA3 or the survival of cells in the presence of Ampicillin
• Regulated yeast promoter (GAL1)
  
  • located upstream of the cDNA insertion site
  • Active in yeast when galactose is present as a carbon source
  • Inactive in presence glucose
  • So activation of the promoter would express the cDNA

Question 9

Week 10

cDNA (Lambda YES) is transformed into yeast strain by electroporation

Answer 9

Week 10

• Goal: cDNA (Lambda YES) is transformed into yeast strain strain S5 (MATa met3, ura3, leu2) by
• cDNA library aliquot provided had a concentration of 4 μg/2 μl. The desired concentration was 0.13 μg/μl
  
  • The following dilution was performed to achieve the final concentration
  • Copy and paste link

https://drive.google.com/file/d/1frR5XNHq2wQyFfIwsrp_3nLQd43WZ-F-/view?usp=sharing

• S5 (MATa met3, ura3, leu2) was selected because
  
  • met3 mutation
    
    • Doesn’t allow yeast to synthesize methionine
    • if yeast is successfully transformed with the higher plant gene (MET3 wild type homolog in A. thaliana), and the plant gene is expressed, then it can substitute the mutant yeast gene, confirming genetic complementation has occurred
  • ura3 mutation
    
    • Used in conjunction with the wild type URA3 gene in the lambda YES vector, which is a selectable marker
    • selects for cells that have incorporated the plasmid
    • denoted by cells that can grow in uracil drop out medium
• transformants plated on sorbitol (glucose), methionine, leucine and no uracil
  
  • uracil not added to select cells that gained the plasmid
    
    • Lambda YES can synthesize it’s own uracil
  • Supplemented with leucine and methionine, required by the yeast
    
    • leu2 mutation not applicable to experiment
  • glucose is used to not allow expression of A. thalian genes
    
    • Inactivates Lambda YES GAL1 promoter
    • We just want to confirm the cells gained the plasmid
    • GAL1 promoter is active in yeast when grown on galactose and is inactive if glucose is used as in this case

Question 10

Week 11

induced expression of all cDNAs

Answer 10

Week 11

• Goal: induced expression of all cDNAs
• Determine transformation efficiency: # of transformants per ug DNA
  
  • olony forming units/ug DNA
  • Typical range is 10² – 10⁴
  • Each transformant is a colony
  • Colonies on 5 and 15 μl were counted to determine transformation efficiency
  • Copy and paste link

https://drive.google.com/file/d/1frR5XNHq2wQyFfIwsrp_3nLQd43WZ-F-/view?usp=sharing

• transformants plated on galactose, methionine, leucine and no uracil.
  
  • uracil not added to select cells that gained the plasmid
    
    • Lambda YES can synthesize it’s own uracil
  • methionine and leucine added because the yeast strain requires both
    
    • leu2 not applicable to experiment
  • galactose is used to allow express of A. thaliana genes
    
    • Lambda YES has GAL1 promoter
    • GAL1 promoter is active in yeast when grown on galactose and is inactive if glucose is used

Week 11 - follow up

• transformants plated on galactose and leucine (no methionine or uracil)
  • leucine added because initial yeast strain is mutant for leu2
    
    • not applicable to experiment
  • uracil is not added to select cells that gained the plasmid
    
    • Lambda YES can synthesize it’s own uracil
  • galactose is used to allow expresson of A. thalian genes
    
    • Lambda YES has GAL1 promoter
    • GAL1 promoter is active in yeast when grown on galactose and is inactive if glucose is used
  • Methionine is not added to select for cells that can now make their own methionine
    
    • They acquired the plant gene homolog of the yeast MET3 gene from the plasmid
    • colonies that grow are likely transformed with a complementing A. thaliana cDNA, which allows the yeast to synthesize methionine
    • colonies that die could be mutants of other dDNA transformations, but die because they did not transform for MET3

Question 11

Week 12

transformants plated on galactose (no methionine)

Answer 11

Goal: transformants plated on galactose (no methionine)

• Select mutant yeast strains expressing the complementing A. thaliana cDNA and re-plate
  
  • All growth denotes acquired the plant gene homolog of the yeast MET3 gene from the plasmid and are expressing a complementing A. thaliana cDNA
• Replated with no methionine to separate the mutants (acquired the plant gene homolog of the yeast MET3 gene from the plasmid) from any other mutants in the prior plate, and grow them to a larger quantity for the next experiment

Question 12

Week 13

Retrieve plasmid from a positive yeast colony

and transform it into E. coli. using electroporation

Answer 12

Goal: Retrieve plasmid (Lambda YES vector with A. thaliana cDNA) from a positive yeast colony and transform it into E. coli. using electroporation

• it is difficult to grow plasmid in yeast because it cannot replicate, but it can in E. coli
• Electroporation used instead of heat shock method cuz of its high efficiency
  
  • were starting w/a small amount of cells needed a higher mass
• E. Coli was plated onto LB + ampicillin plates
  
  • selects transformed cells
  • Because E. coli can grow on all the media we used to plate yeasts in this experiment, and as a result we need a new way to select for transformants
  • we took advantage of the ampicillin resistance selectable marker of the plasmid, to select for transformants of E. coli
  • We couldn’t use ampicillin for the selection of yeast because S. cerevisiae are naturally resistant to ampicillin.
• 2 controls were also transformed
  
  • Control plasmid transformed into E. coli
    
    • A known amount of a control plasmid was used to determine that E. coli is competent (able to accept foreign DNA)
    • competency is confirmed if E. coli can grow on LB + amp plates
    • If it can grow in the presence of amp, then it was transformed with Lambda YES (which has the ampicillin resistance gene)
    • helps troubleshoot errors
      
      • Since the plasmid isolated from the yeast was not quantitated because there is not enough to measure, you cannot calculate the transformation efficiency of your plates
      • Two reasons why you may not generate colonies in your plates
        
        • Plasmid was not isolated from the yeast colony
        • E.coli cells used for transformation were bad
  • Control w/no DNA
    
    • Bacteria was electroporated and no plasmid was used during transformation, then the bacteria was plated on LB + ampicillin
    • No growth was expected from this control
    • Confirms that the addition of the plasmid is what gives the bacteria the selectable marker, which is ampicillin resistance
• determining transformation efficiency of the control plasmid can help you figure out what went wrong
  
  • Electrocompetent E. coli cells should have a transformation efficiency at least 1 x 10⁸ colonies/ ug DNA.
  • If electrocompetent cells are good, and there were no colonies with the sample, then that suggests there is a problem with the yeast sample yeast plasmid extraction will need to be redone
  • If transformation efficiency is less then expected then electrocompetent cells are not good, and a new batch of E. coli is needed to the E. coli transformation
• Transformation efficiency of control sample was calculated
  
  • transformation efficiency is colonies per ug of DNA
  • Copy and paste link

https://drive.google.com/file/d/1frR5XNHq2wQyFfIwsrp_3nLQd43WZ-F-/view?usp=sharing

• Once grown in E. coli, it will be isolated
  
  • Plasmid inserted into yeast to confirm phenotype (if we had time, but we don’t so skip this step)

Question 13

Week 14

isolate the plasmid from an E. coli transformant

Answer 13

• isolate the plasmid from an E. coli transformant using a plasmid prep kit
• plasmid should have the Arabidopsis MET3 gene as the cDNA
• determined the concentration of the plasmid, and based on that the total yield to confirm purity of DNA
  
  • how much DNA was recovered in the plasmid prep
  • Copy and paste link

https://drive.google.com/file/d/1frR5XNHq2wQyFfIwsrp_3nLQd43WZ-F-/view?usp=sharing

• Once confirmed, different restriction digests were used for DNA fragment separation via electrophoresis.
• Fragment sizes per restriction digests were used to approximate clone size and generate a restriction map
• Electrophoresis results were used in conjunction with the Lambda YES map provided to generate the restriction map
• clone size was approximated to be 2000 bp and a restriction map was generated
• According to the gel digest and the restriction map generated, the Lambda YES plasmid was successfully isolated, and the cDNA library was located within the plasmid
• The location of the cDNA was as expected
• results validated that the attempt to clone a higher plant gene by complementation of a yeast strain was successful

Question 14

Calculations

Answer 14

https://drive.google.com/file/d/1frR5XNHq2wQyFfIwsrp_3nLQd43WZ-F-/view?usp=sharing