Part:BBa_K2310100:Experience

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Applications of BBa_K2310100

Experimental objective

This experiment is designed to explore the influence of IPTG induction dosage, induction time, induction temperature, substrate dosage, microbial concentration (OD600 value), and other conditions to the chemiluminescence of luxAB luciferase.

Induction temperature

We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter+luxAB (BBa_K2310103)in the fluid medium. When the value of OD600 reached about 1.2, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced at 30℃, 33℃ and 37℃, 190rpm shaking table for 3 hours respectively. Took 1ml bacteria solution in an EP tube, added 5ul substrate capraldehyde into EP tubes respectively and then took 100ulinto 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.

Figure 1. Induction temperature –standard luminescence value

(Note: standard luminescence value= luminescence value-background value)

We also did the experiment again under the same condition and induced in 30℃ and 37℃ for 3 hours.

Figure 2. Induction temperature –standard luminescence value

(Note: standard luminescence value= luminescence value-background value)

From the diagram, we can draw the conclusion that 30℃ is the best induction temperature among these three induction temperatures.

IPTG induction dosage

We cultured the DH5a bacteria which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul, 8ul, 12ul, 16ul, 20ul IPTG in the tubes respectively. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.

Figure 3. IPTG induction dosage-relative luminescence intensity

(Note1: relative luminescence intensity= luminescence value/background value)

(Note2: The relationship between the lines is incommensurable)

The diagram shows that when OD600 is low, the relative luminescence intensity will rise with the increase of the IPTG induction dosage within a certain range. However, when OD600 is high, the relative luminescence intensity may not rise with the increase of the IPTG induction dosage because the IPTG dosage is nearly saturated. By the way, 1.88 may be the best value of OD600 to test the influence of IPTG induction dosage. We guess that these two factors have an exponential function when OD600 is low; so we try to fit a curve under the first three values of OD600.

Figure 4. IPTG induction dosage-relative luminescence intensity while OD600=0.8

Figure 5. IPTG induction dosage-relative luminescence intensity while OD600=1.24

Figure 6. IPTG induction dosage-relative luminescence intensity while OD600=1.88

Microbial concentration (OD600 value)

We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.

Figure 7. OD600 value-relative luminescence intensity

(Note: relative luminescence intensity= luminescence value/background value)

The diagram shows that the relative luminescence intensity will rise with the increase of OD600 value within a certain range. Besides, it seems that these two factors have an exponential function; as a result we try to fit a curve under different IPTG induction dosages.

Figure 8(a). OD600 value-relative luminescence intensity (1/k IPTG)

Figure 8(b). OD600 value-relative luminescence intensity (2/k IPTG)

Figure 8(c). OD600 value-relative luminescence intensity (3/k IPTG)

Figure 8(d). OD600 value-relative luminescence intensity (4/k IPTG)

Figure 8(e). OD600 value-relative luminescence intensity (5/k IPTG)

When the concentration of IPTG is low, the exponential function between OD600 value and relative luminescence intensity is significant relatively. However, when the concentration of IPTG is high, the function is not significant. This is also because that the IPTG dosage is saturated relatively for the high OD600 value.

Induction time

We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced1, 2, 3 hours in 30℃, 190rpm respectively, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.

Figure 2.2.9Induction time-relative luminescence intensity

(Note1: relative luminescence intensity= luminescence value/background value)

(Note2: The relationship between the lines is incommensurable)

This group of data is a bit odd; we cannot say the relationship between the induction time and relative luminescence intensity, so we tested the data in a different way.

Figure 10. Induction time-relative luminescence intensity value

(Note1: relative luminescence value= (luminescence value-background value)/OD600 value after induction)

(Note2: The relationship between the lines is incommensurable)

The diagram shows that the induction time has little influence on the relative luminescence value. Consequently, we can choose 1 hour as our induction time in order to improve the efficiency.

Substrate dosage

We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteriasolution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 5ul, 10ul, 15ul, 20ul substrate capraldehyde into EP tubes respectively and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.

Figure 2.2.1Substrate dosage-relative luminescence intensity

(Note1: relative luminescence intensity= luminescence value/background value)

(Note2: The relationship between the lines is incommensurable)

The diagram shows that when OD600 is low, the influence of substrate dosage on relative luminescence intensity is minimal. However, when OD600 is high, the relative luminescence intensity will rise with the increase of the substrate dosage within some certain range because of the accumulation of the luxAB luciferase.

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