Choice of strains and plasmids

Choice of strains and plasmids

Choice of strains and plasmids

There are two strains of yeast were used in this project,known as wild-type strain (YN94-1) and the mutated strain (YN97-147)where the strain has been mutated in mismatch pair protein, recognized asMsh3. A high-copy 2-μ based vector plasmid;known as pKa3- 9(AC)41(A)2(C)2(AC)4(A) was used according to the method stated in section 2.4.1. This plasmid was chosen due to its long repetitive tract, which can be a source of the DNA damage. Apart from that, the plasmid, pKa3-9(AC)41(A)2(C)2(AC)4(A) provide a qualitative assay. It is hypothesized that the frequency of frame slippage of the wild-type (YN94-1) strain will be lower than the mutated strain (YN97-147msh3Δ).

The transformants were initially grown on SC-U, and then incubated for 2 days at 30°C. From the selected plates, three colonies were chosen (labelled A, B, C) and restreaked onto fresh SC-U plates and incubated for 2 days at 30°C. Single yeast colonies were picked from A, B and C and inoculated individually into 10 ml SC-U in universal bottles and incubated in the rotary shaker overnight at 170rpm at 30°C. The number of cells/ml in the overnight culture was determined spectrophotometrically at a wave length of 600 nm (1 OD = 3 x 107cells). Serial dilutions were carried out and specific aliquots were plated out onto SC-U plates (control plates) and SC-U plates with added known volumes of tea. These plates were left to incubate for 2 days at 30°C.

The numbers of colonies grown were counted(Table 3.1). For pure green tea experiments, there were sufficient number of colonies per plate (300-500) for both yeast transformants (YN94-1 WT [pKa3-9(AC)41(A)2(C)2(AC)4 (A)] and YN97-147 Δmsh3[pKa3-9(AC)41(A)2(C)2(AC)4(A)]). However, there was far less than expected colonies per plate(120- 250) for the most experiments of green tea with mint, even though everything had done as the same procedures as the pure green tea experiments (the raw data 7.1.1 of the appendix). The reason for this is uncertain. It is possible that errors had occurred during the transformation, which resulted in some of the cells not being transformed successfully.

Table3.1Numbersof coloniesperplate

Average CFU/plates ±SEM
Green tea+ mint     Pure green tea
Strains     0ml     50ml     100ml     0ml     50ml     100ml
YN94-1WT[pKa3-9(AC)41(A)2(C)2(AC)4(A)]     203.8± 61

182.0± 40

175.4± 73

431.8± 91

350.8± 11

331.6± 58

YN97-147msh3Δ[pKa3-9(AC)41(A)2(C)2(AC)4(A)]     183.6± 89

213.2± 83

166.2± 64

370.6± 66

326.6± 52

314.0± 50

The table shows the  average number of colonies  per plate±SEM.

After the colonies have grown,theβ-galactosidaseoverlayassayisconducted.The basis of theβ-galactosidaseoverlayistoobservethecolourtransitionofthecoloniesfrom white to blue. White colonies represent the ‘out-of-frame’ assayplasmid (Mardenetal,2006) whereas the blue colonies represent the frame slippage event. Green tea with mint plates had converted quickly from white to blue. However,for pure green tea,the colonies were mostly pink in colour.This makes it difficult to distinguish the frame slippage events. Some colonies were found to be pink with slight blue tinge in the middle or   colonies underwent as light colour change which was not quite blue. Subjective judgement had to be made as to whether to count these colonies as frame slippage events.

3.1.2 Choice and volumes of tea

The teas used in this study are green tea with mint and pure green tea. From previous studies, it was found that green tea has an ability to reduce DNA damage. However, tests with green tea with flavourings have not been conducted before, so it is not known how these flavourings would affect yeast growth and theβ-galactosidase assay.

Both types of tea were prepared similarly before addition to SC-U media. Two tea bags (weighing approximately 2g per bag) were infused in 200ml of boiled distilled water for 3 minutes with continuous stirring. This gave a 2% (w/v) concentration. Two different concentrations of both green tea with mint and pure green tea were done in this study (25/50 ml and 50/100 ml). Firstly, 50 ml and 100 ml of the teas were added to 450 ml and 400 ml SC-U media respectively to make 500 ml before being poured onto plates. For the lower concentration (25/50 ml), 25 ml and 50 were added to 450 ml and 400 ml SC-U respectively. streak

The control plates and SC-U plates containing the different concentration of tea were streaked with specific amount of aliquots of all four transformants. A sufficient amount of colonies was able to grow on the plates for both volumes of pure green tea (about 300-700 colonies) while there were less colonies of the most green tea with mint experiments (120-250). The raw data of the whole experiments are in table 7.1.2 of the appendix.
3.2 The ability ofgreen teawith mint to reduce DNA damage

The yeast transformants, YN94-1WT[pKa3-9(AC)41(A)2(C)2(AC)4(A)],YN97-147msh3Δ[pKa3-9(AC)41(A)2(C)2(AC)4(A)] wereplatedonto5control plates(SC-Uonly),5platesoftheSC-Uwith50mland100mlgreenteawith mint. After incubation for two days, the plates were overlaid for theβ-galactosidaseassay(MaterialsandMethods2.4.3) and left on the bench for 7-14 days for colour development to occur. The number of blue colonies was counted and the frequency of blue colonies against the total number of colonies on the plates was calculated.The results from the experiments are shown graphically in Figures 3.1and3.2.

Figure3.1showstheabilityofgreenteawithmintto reduce DNA damage in the wildtypestrainandthemsh3Δmutatedstraintransformedwiththeplasmid,pKa3-

9(AC)41(A)2(C)2(AC)4(A).Thebargraphillustratesthatgreenteawithminthasthe abilitytoreducethenumberofbluecolonies, whichisanindicationthatDAN damageisincreased.Thenumberofbluecoloniesreducesasthevolumeofthetea increases.Inthewildtypestrain, YN94-1WT[pKa3-9(AC)41(A)2(C)2(AC)4(A)],the relativefrequencyofbluecoloniesdecrease43%and83%when25mland50mlof theteawereused,respectively.Inthemutatedstrain, YN97-147msh3Δ[pKa3-9(AC)41(A)2(C)2 (AC)4(A)],thereisadecreaseof90%and95%.Forbothstrains, therearesignificantdifferences(p<0.05)betweenthecontrolplatesandthe25ml and50mlgreenteawithmintplates.Hence,provingthattheadditionofgreentea withmintplaysapositiveroleinthepromotingmicrosatellitestabilityinyeastDNA. Therawdatafromtheseexperimentsareshownin Tables7.1.1and7.1.2ofthe Appendix.
Figure3.1         Theabilityofgreenteawithmint to reduce DNA damageintheYN94-1 wild typeandYN97-147msh3ΔstrainswithpKa3-9(AC)41(A)2(C)2(AC)4(A60
50

40

*

30

20

*

10

*                                       *

0

0ml                                   25ml                                   50ml

Volumesof GreenTeawithmint addedtoSC-U

Thebargraphshowstheability of greenteawithmint to reduce DNA damagein theYN94-1wildtypeand YN97-147msh3Δstrainswith pKa3-9(AC)41(A)2(C)2(AC)4(A). The columnsrepresentthe relative frequencyofblue colonies(multipliedby 100)andtheerrorbarsrepresentthestandarderrorofmean (SEM).Significantdifferencesinthedecreaseofframeslippagecanbeseeninboth strains(denoted by*= p<0.05).
3.3abilityof pure green tea to reduce DNA damage

ThesamestrainscontainingthesameplasmidswereusedontheSC-Uplateswith pure greentea.Thevolumesofthe puregreentea were50ml and 100ml.The number of blue colonies was countedaftertheβ-galactosidaseoverlayhasbeenconducted.Table3.2showsthenumberofbluecoloniesfoundin thecontrolplatesandSC-U containing 25 ml and50ml of pure greentea.

Table3.3Theabilityof pure greenteato reduce DNA damage.

β-Galactosidaseexpression± SEM
Strains     0ml     50ml     100ml
YN94-1WT

[pKa3-9(AC)41(A)2(C)2(AC)4(A)]
4.2×10-1 ± 7.9×10-2     6.0x 10-3± 5.7x 10-3*     6.0x 10-3±5.7x 10-3
YN97-147msh3Δ

[pKa3-9(AC)41(A)2(C)2(AC)4(A)]
1.9×10-1 ± 4.1×10-2     8.0×10-4±5.6×10-4*     2.3x 10-4± 1.5x 10-4

Thevaluesshownarethe relativefrequencyofbluecolonies± SEM (standarderrorof mean), where* denotes p<0.05

Fromtheresultsshownabove,therelativefrequencyofbluecoloniesfortheplates containing50 mland100 mlofteawasverylowincomparisontothe controlplatesaswellastheplatescontaining greenteawithmint.Therawdatafromtheseexperiments areshowninTables7.2.1,7.2.2,7.2.3and7.2.4 oftheAppendix.

Itwasunknownthelowrelativefrequencyofbluecoloniesisduetothe effectivenessofthegreenteainpreventingDNA damageorbecauseerrors had happenedduring theβ- galactosidaseoverlayassay.Hence,arepeatexperimentwasconductedwithallthe strainstoseeifbluecoloniescanbeexpressedinloweredvolumesofteainSC-U plates.The volumesofthegreenteawerereducedto25mland50ml. TheresultsoftherepeatexperimentscanbeseeninFigures3.3and3.4forallthe strains.

Figure3.3showsthe abilityofgreenteato reduce DNA damage in thewildtype strainandthemsh3Δmutatedstraintransformedwiththeplasmid, pKa3-9(AC)41(A)2(C)2(AC)4(A) atlowervolumesof25 ml and50 ml.Thegraphshows

adecreasing trend in relative frequency of blue colonies as the volume of tea

26

increases.Inthewildtypestrain,YN94-1WT[pKa3-9(AC)41(A)2(C)2(AC)4(A)],the relativefrequencyofbluecoloniesdecreasedto26%inthe25mlteaplatewhereas inthe50mlplate, there wasa58%decrease.Inthemutatedstrains,YN97-147msh3Δ [pKa3-9(AC)41(A)2(C)2(AC)4(A)],therewasadecreaseof88%and94%for the25mland50mlplatesrespectively.Theraw datafromthese experimentsareshowninTable7.2.5oftheAppendix.

Figure3.3         Theabilityofgreenteatoreduce DNA damage inwildtype YN94-1and YN97-1msh3ΔstrainswithpKa3-9(AC)41(A)2(C)2(AC)4(A) usinglower volumesof 25ml and50mlof greentea.

100

90

80

70

60

50

40

30

20

10

0

0ml                                25ml                               50ml

Volumesof GreenTeaaddedto SC-U

The graph shows the ability of green tea to reduce DNA damage intheYN94-1wildtypeand YN97-147msh3Δstrainswith pKa3-9(AC)41(A)2(C)2(AC)4(A). The columns represent the relative frequency of blue colonies(multiplied by 100)and the error bars represent the standard error of mean (SEM). There is no significant  difference.