Team:XMU-China/Results
In our experiment, for testing the amplification effect of our circuit, we used GFP as the reporter, because the fluorescence intensity is easier to be measured. Also, the intensity of the fluorescence can stand for the intensity of the corresponding genes' expression, and the data of the fluorescence intensity could be more convenient for us to model the circuit. Meanwhile, we chose BBa_J33201, an arsenic induced promoter, as our sensor to detect arsenic in water samples.
Figure 1.1 The amplifier test system (Experimental group)
Figure 1.2 The no-amplified system (Control group)
We designed a double-plasmid system, which contains BBa_K2310009 and BBa_K2310002 as Figure 1.1 shows, and a control group that only contains a GFP expression system with the same promoter that can be induced by arsenic as Figure 1.2 shows. Both these two systems were transformed into E.coli DH5 and incubated with arsenious acid standard solution (the concentration of arsenic is 0, 2.6, 6.5 and 97.5 μM) for 24 hours. After that, we measured OD600 of the E.coli and the fluorescence intensity of GFP for 900 minutes. The OD600 showed the growth trend of the engineering bacteria and the fluorescence intensity of each group showed the relationship between arsenic and the expression of our circuits.
To ensure that the concentration of arsenic would not influence the growth of the bacteria, we incubated our bacteria with adding arsenious acid standard solution into the medium.
Figure 1.3 The OD600 versus time of E.coli with different concentration of arsenic
From Figure 1.3, we found that OD600 after incubating for 24 hours is about 0.55, and the growth curves of each group don't show much difference. It suggested that they had the same growth trend, so the difference of fluorescence intensity was mainly from the expression of the circuits or other factors.
Figure 1.4 The average green fluorescence intensity versus time of each group
Then we did experiments at the same condition to measure the fluorescence intensity of GFP. As showed in Figure 1.4 with the increase of arsenic, the expression of the GFP showed an upper trend. In addition, the green fluorescence intensity of the amplified group (showed in Figure 1.1) is much higher than that of the unamplified groups (showed in Figure 1.2), about 1.5 times higher.
These show that the amplification effect of our circuit is effective and all right, and our system works well.
I. The function of lacZ and electrochemical detection
Firstly, our constructed BioBrick, BBa_K2310003 was transformed into E.coli BL21(DE3) and incubated on X-gal-IPTG plate overnight. After a 20 hours incubation, blue-white colonies can be observed on the plate.
Figure 2.1.1 Blue-white colonies on X-gal-IPTG plate
Secondly, we chose a single colony with blue-white color and incubated in 10mL LB fluid medium overnight. To ensure our BioBrick works well, we re-incubated some medium on X-gal-IPTG plates, with BBa_K2310002 and BBa_K2310004. After about four hours, the color of colonies changed (as Figure 2.1.2 shows), so we can confirm that our BioBricks can work normally.
Figure 2.1.2 Re-incubated medium on X-gal-IPTG plates
Then, to produce β-galactosidase quantificationally, the medium of BBa_K2310003, was divided into 4 groups equally, and induced by IPTG. The concentration of IPTG in each group is 0, 0.01, 0.05, 0.1 mM. After adding IPTG solution into the medium, we incubated them for another 45 minutes.
After the incubation, we measured the OD600 of the medium, separated the bacteria by centrifuge and resuspended with PBS. Then, PAPG solution was added, and the bacteria were incubated at 37 for another 30 minutes. Finally, the liquid was tested by cyclic voltammetry on an electrochemical workstation with a three-electrode system.
At the same time, another groups with T7 promoter and RFP (BBa_K2310104) were also induced by IPTG at the same condition, and the red fluorescence was measured for a contrast.
Figure 2.1.3 The relationship of OD600 and the concentration of IPTG
From Figure 2.1.3 we can learn that the growth of our bacteria could be influenced by IPTG when the concentration of IPTG is high, because of the toxicity of IPTG. But when IPTG is at a low concentration, the influence of IPTG can be ignored.
Figure 2.1.4 The relationship of OD600 and the concentration of IPTG
Figure 2.1.4 shows that when the concentration of IPTG increases to 0.1mM, the growth of bacteria will be influenced, but when the concentration of IPTG is not too high, there would be a proportional relation between the concentration of IPTG and the fluorescence intensity. In the other hand, the fluorescence can be detected after 2 hours.
Figure 2.1.5 The Potential-current curve of c(IPTG)=0
From Figure 2.1.5 we can learn that when there's no IPTG in the system, lacZ would not express, so there's no β-galactosidase in the system. Meanwhile, the electrochemical analysis will show the electrochemical property of PAPG and the curve can be used as a standard curve. From the reference we can know that while there's PAP in the system, the current at the potential of ~-0.2V will be stronger, and the strength of the current is in direct proportion to the concentration of PAP. So, when there's no PAP in the system, the current is about 6.5e-5A.
Figure 2.1.6 The Potential-current curve of c(IPTG)=0.01mM
In Figure 2.1.6 when the concentration of IPTG is 0.01 mM, the current at the potential of ~-0.2V rises to about 8.5e-5A.
Figure 2.1.7 The Potential-current curve of c(IPTG)=0.05mM
In Figure 2.1.7, when the concentration of IPTG is 0.05mM, the current at the potential of ~-0.2V rises to about 9.5e-5A, higher than that at the concentration of IPTG is 0.01 mM.
Figure 2.1.8 The Potential-current curve of c(IPTG)=0.1mM
In Figure 2.1.8, when the concentration of IPTG is 0.1mM, the current at the potential of ~-0.2V rise to about 6.5e-5A, even the same as that at the concentration of 0mM. Combined with our conclusion of the influence of IPTG to bacteria cells, we can draw three conclusions:
1. There's a proportional relation between the concentration of IPTG and the strength of current while the concentration of IPTG is lower than 0.1 mM.
2. The measure of our electrochemical analysis with T7 expression system can be really sensitive and fast, only a total of 75 minutes incubation would be needed, but the fluorescence protein needs at least 2 hours to produce a signal that can be detected.
3. Our electrochemical analysis method with lacZ works well.
II. The function of LuxAB
1. 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.
2. 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 4μL IPTG in the tubes. Induced in 30℃, 33℃ and 37℃, 190rpm shaking table for 3 hours respectively. Took 1ml bacteria solution in an EP tube, added 5μL substrate capraldehyde into EP tubes respectively and then took 100μL into 96-well plates to measure the luminescence. We also took 100μL bacteria solution to measure the value without capraldehyde.
Figure 2.2.1 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 2.2.2 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.
3. 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 4μL, 8μL, 12μL, 16μL, 20μL 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 10μL substrate capraldehyde into EP tubes and then took 100μL into 96-well plates to measure the luminescence. We also took 100μL bacteria solution to measure the value without capraldehyde.
Figure 2.2.3 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 2.2.4 IPTG induction dosage-relative luminescence intensity while OD600=0.68
Figure 2.2.5 IPTG induction dosage-relative luminescence intensity while OD600=1.24
Figure 2.2.6 IPTG induction dosage-relative luminescence intensity while OD600=1.88
4. Microbial concentration (OD600 value)
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 bacteria solution in the glass test tubes and added 4μL IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10μL substrate capraldehyde into EP tubes and then took 100μL into 96-well plates to measure the luminescence. We also took 100μL bacteria solution to measure the value without capraldehyde.
Figure 2.2.7 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 2.2.8(a) OD600 value-relative luminescence intensity (1/k IPTG)
Figure 2.2.8(b) OD600 value-relative luminescence intensity (2/k IPTG)
Figure 2.2.8(c) OD600 value-relative luminescence intensity (3/k IPTG)
Figure 2.2.8(d) OD600 value-relative luminescence intensity (4/k IPTG)
Figure 2.2.8(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.
5. 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 bacteriasolution in the glass test tubes and added 4μL IPTG in the tubes. Induced 1, 2, 3 hours in 30℃, 190rpm respectively, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10μL substrate capraldehyde into EP tubes and then took 100μL into 96-well plates to measure the luminescence. We also took 100μL bacteria solution to measure the value without capraldehyde.
Figure 2.2.9 Induction 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 2.2.10 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.
6. 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 bacteria solution in the glass test tubes and added 4μL IPTG in the tubes. Induced 3 hours in 30℃, 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 5μL, 10μL, 15μL, 20μL substrate capraldehyde into EP tubes respectively and then took 100μL into 96-well plates to measure the luminescence. We also took 100μL bacteria solution to measure the value without capraldehyde.
Figure 2.2.1 Substrate 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.
Xiamen University, Fujian, China
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