Calculations for Mutagenesis Lab

Variables:CO: Original concentration of cellsCV: Concentration of viable cellsD: Dilution spread for original dilutionS0: # of colonies on the un-irradiated minimal medium + histidine plateSi: # of cells initially placed on the minimal medium + histidine platesSt: # of colonies on the minimal medium + histidine plates at time tM0: # of colonies on the un-irradiated minimal medium plateMi: # of cells initially placed on the minimal medium platesMt: # of colonies on the minimal medium plates at time tDs: Dilution factor for the minimal medium + histidine platesDM: Dilution factor for the minimal medium platesNS: # of viable cells for the minimal medium + histidine plates before UV

exposure/irradiationNM: # of viable cells for the minimal medium plates before UV

exposure/radiationPt: Portion of survivors for the minimal medium + histidine plates at time tFS: Frequency of spontaneous reversions in the minimal medium platesRst: # of spontaneous reversions in the minimal medium plates at time tRit: # of induced reversions in the minimal medium plates at time tFit: Frequency of induced reversions in the minimal medium plates at time t

Formulas:

CO=¿colonies

D

COaverage=DCO

¿ plates

CV=S0Ds´ 10

DM=M i

CV

DS=SiCV

N S=CV ´ DS ´ 0 .1mLNM=CV ´ DS ´ 0 .1mL

Pt=S tS0

FS=M 0

NMRst=P t ´ M 0

Rit=M t−Rs t

Fit=Rit

NM ´ P t

# of colonies on minimal medium + histidine (MM+) plates: 10-6 dilution: 45 (plate 1) and 35 (plate 2) 10-7 dilution: 3 (plate 1) and 2 (plate 2)

Original Concentration CO values:

4.2x108cells/mL (10-6 plate 1)

3.5x108cells/mL (10-6 plate 2)

3x108cells/mL (10-7 plate 1)

2x108cells/mL (10-7 plate 2)

Sample calculation for CO on 10-6 dilution plate 1:

CO=¿colonies

D´ 10=45cells

106´ 10=4 .5 ´ 108cells /mL

Sample calculation for CO average:

COaverage=DCO

¿ plates=4 .2´ 10

8+3 .5 ´ 108+3 ´ 1082 ´ 108

4=3.25 ´ 108cells /mL

CV is the number of cells from the original solution that were viable. The sample calculation for CV is:

CV=S0Ds´ 10=151cells

10−6 ´ 10=1 .51 ´ 109 cells

This CV value is much greater than CO although it should be smaller. This is because the standard MM+ plates were used for the results, where the CO was not given *** THIS SCREWS UP ALL OUR STUFFS CUZ THERES MORE VIABLE CELLS THAN THERE WERE CELLS TO BEGIN WITH OMGGGGGGGG

Because we want 30 cells to spontaneously revert per 100µL on the minimal medium (MM) plates (should have 10-6 frequency, or one in one million), we want 30x106=3x107 cells per 100µL (3x108 cells per 1mL).Sample calculation for the dilution factor for the MM plates (DM):

CV=M i

DM´ 10→DM=

Mi

CV= 3 .0 ´ 108

1.51´ 109´ 10@1

Unfortunately this theoretical dilution factor is not possible, as there is only 100µL of cells remaining, and there must be a dilution of 10-1 in order to have enough solution to spread 100µL on the 7 plates required.

To calculate the DS value, we want 300 cells per 100µL (3000 cells per 1mL) to be on the minimal medium + histidine (MM+) plates before irradiation.Sample calculation for DS:

CV=SiDS´ 10→DS=

SiCV

=30001.51´ 109

´ 10@10−4

The results of this dilution produced plates with too many colonies to count with the method used; therefore the results from the standard plates were used. The standard plates had a dilution factor of 10-6.

Sample calculation for # of viable cells for each MM+ plate before UV light exposure/irradiation:

N S=CV ´ DS ´ 0 .1mL=(1 .51´ 109cells /mL)´ (10−6 )´ 0.1mL=151cells

Sample calculation for # of viable cells for each MM plate before UV light exposure/irradiation:

NM=CV ´ DS ´ 0 .1mL=(3 .25 ´ 108 cells /mL)´ (10−1 )´ 0 .1mL=1 .51 ´ 107 cells

Pt values:P0 sec = 1.00P2 sec = 0.993P5 sec = 0.795P7 sec = 0.742P10 sec = 0.702P20 sec = 0.205P30 sec = 0.0662

Sample calculation of the proportion of survivors at time t for MM+ plates:

Pt=S tS0

→P10 sec=S10secS0

=106cells151cells

=0 .702

Sample calculation of the frequency of spontaneous reversions in the un-irradiated MM plate:

FS=M 0

NM=42cells1 .51 ´ 107cells

=2 .78 ´ 10−6

Number of spontaneous reversions in the MM plates (Rst) at time t:Rs0 sec = 42 cellsRs2 sec = 42 cellsRs5 sec = 33 cellsRs7 sec = 31 cellsRs10 sec = 29 cellsRs20 sec = 9 cellsRs30 sec = 3 cells

Sample calculation for # of spontaneous reversions in the MM plates:

Rst=P t ´ M 0→Rs10 sec=P10sec ´ M 0=0 .702 ´ 42cells=29cells

Number of induced reversions in the MM plates (Rit) at time t:Ri0 sec = 0 cellsRi2 sec = 124 cellsRi5 sec = 357 cellsRi7 sec = 510 cellsRi10 sec = 741 cellsRi20 sec = 535 cellsRi30 sec = 247 cells

Sample calculation for Rit at t10 seconds:

Rit=M t−Rs t→Ri10 sec=M 10sec−Rs10 sec=770 cells−29cells=741cells

Frequency of induced inversions (Fit) at time t:Fi0 sec = 0Fi2 sec = 8.27x10-6

Fi5 sec = 2.98x10-5

Fi7 sec = 4.55x10-5

Fi10 sec = 6.99x10-5

Fi20 sec = 1.73x10-4

Fi30 sec = 2.47x10-4

Sample calculation for Fit for MM plates at 10 seconds:

Fit=Rit

NM ´ P t→Fi10 sec=

Ri10 secNM ´ P10sec

=741cells(1 .51 ´ 107 cells)´ (0.702)

=6 .99 ´ 10−5