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# ING - ING-Induction Assay and Electrophoresis Name Nick...

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1 ING -Induction, Assay and Electrophoresis Name: Nick Benzschawel Partner: Tony Desotell Section: 301

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2 Induction Experiments 1. Plot of growth curves Figure. 1. Growth curve for each culture constructed from DATA SHEET II. Since OD 410 =0.15 corresponds to a cell concentration of 10 8 cells. In order to calculate the number of cells per milliliter in a particular concentration, one must take the OD 410 value for that culture and multiply it by the factor 10 8 to convert the value to the appropriate scale, and then divide this value by 0.15 to get the approximate cell concentration. Sample calculation for Cells/mL : Generic equation for calculating cell concentration (Cells/mL): OD 410 10 8 0.15 Cells/mL Sample calculation using 20min Control value: 0.135 10 8 0.15 9.0 10 7 Cells/mL
3 2. Calculation of culture doubling times Figure 2. Plot ln[A 410 /A 0 ] versus incubation time. The increasing order of doubling time was lactose ( T D =52.51 min), IPTG ( T D =55.41 min), control ( T D =77.88 min), antibiotics ( T D =1386.29 min). The cell cultures treated with the antibiotic chloramphenicol had the longest doubling time while the cells treated with the gratuitous inducer, IPTG, and the natural inducer, lactose, had the fastest doubling times, which is consistent with intuitive predictions that growth of cells should be inhibited by antibiotic treatment and stimulated by IPTG and lactose, the inducers of the lac operon. Interestingly, the natural inducer had a faster double time than the gratuitous inducer. This may be the result of a larger initial population in the lactose culture than in the IPTG culture. The doubling time for a culture would yield an A 410 /A 0 ratio equal to 2, and the ln(A 410 /A 0 ) would therefore be the ln2. To calculate the doubling time from this information one must obtain the slope for a particular data set from the linear fit of the data. Equating the slope, m, and the value of the doubling time, denoted as T D , to the ln2 and solving for T D yields the doubling time for a culture. NOTE: For consistency checks, notice that the value of T D should have units of time. This is indeed the case since the slopes of these data sets have units of min -1 so dividing the ln2 by the slope yields a value with units of min.

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4 Sample calculation for doubling time : Generic equation for calculating the doubling time (min): ln(A 410 /A 410 ) ln2 T D slope ( m ) T D ln2 slope ( m ) Sample calculation using IPTG slope: T D ln2 0.0125 55.45 min 3. Enzymatic activity calculations Sample calculations for number of mol of p -nitrophenolate, I.U./mL of culture, I.U./10 8 cells : NOTE : All sample calculations correspond to the 50 min lactose data point. 1. Number of mol of p -nitrophenolate Generic equations for determination of mol of p nitrophenolate : A b c c A b V Total 10 6 mol 1 mol mol of p nitrophenolate Where the path length, b , is equal to 1cm, and the molar absorptivity, V Total is the total volume (3mL) of culture (1mL) and suspension reagent (2mL).
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ING - ING-Induction Assay and Electrophoresis Name Nick...

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