LacZ gene is widely used in gene expression regulation in genetic engineering. LacZ gene encoding β-galactosidase (referred to as β-gal) is composed of four subunits of tetramer, can catalyze the hydrolysis of lactose. Beta-gal is relatively stable, with X-Gal as the substrate colored blue, easy to detect and observe.
However, lacZ will not be used just for blue-white selections. Another substrate, PAPG, as figure 1.1 shows, can also be hydrolyzed by β-galactosidase, and one of the hydrolyzete, PAP, a small molecule which can be electrolyzed will be produced. This hydrolysis reaction is very efficient that only need a 30 minutes incubation at 37℃.
Figure 1.1 The mechanism of the production of electrochemical signal
To analyze the concentration of PAP, an electrochemical analysis will be took by cyclic voltammetry on an electrochemical workstation with a three-electrode system.
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 1.2 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 1.2 shows), so we can confirm that our BioBricks can work normally.
Figure 1.3 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 1.4 The relationship of OD600 and the concentration of IPTG
Figure 1.4 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 1.5 The relationship of OD600 and the concentration of IPTG
Figure 1.5 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 1.6 The Potential-current curve of c(IPTG)=0
From Figure 1.6 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 PAG 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 PAG. So, when there's no PAG in the system, the current is about 6.5e-5A.
Figure 1.7 The Potential-current curve of c(IPTG)=0.01mM
In Figure 1.7 when the concentration of IPTG is 0.01 mM, the current at the potential of ~-0.2V rises to about 8.5e-5A.
Figure 1.8 The Potential-current curve of c(IPTG)=0.05mM
In Figure 1.8, 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 1.9 The Potential-current curve of c(IPTG)=0.1mM
In Figure 1.9, 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 two 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.
As we all know, promoter is an important part in the expression of a gene, especially in synthetic biology. For most prokaryotic expression system, negative feedback regulation is more common than positive feedback regulation, one reason is that it is difficult for prokaryote to have an exact positive feedback regulation, for example, many inducible promoters may have a leakage, that the downstream gene can express without being induced. During our project, we found that our bio-amplifier can amplify the leakage of the promoter, so it can give us a new method to judge whether a promoter is a strict inducible promoter or not. Meanwhile, we decide to set up a value which can represent the level of the leakage of an inducible promoter.
I. Method of judging a promoter
For most inducible promoters, their strength is in direct proportion to the concentration of their inducers, this means that although an inducible promoter may have a leakage, when there’s no inducer, the leakage can be very weak to be detected. To solve this problem, we need to enlarge the expression of the downstream gene without adding inducer, and the best solution is using our bio-amplifier.
The formaldehyde induced promoter, BBa_K1334002 is the first one to be judged by our bio-amplifier.
Figure 2.1 Fluorescence intensity of each amplified group at different concentration of formaldehyde
Figure 2.2 Fluorescence intensity of the un-amplified group and the amplified group without formaldehyde
From figure 2.1 we can learn that for each amplified group, the difference between them is not so obvious, and figure 2.2 shows that the fluorescence intensity of amplified group is always stronger than the un-amplified groups, it means that there must be a leakage in this promoter.
From these experiments, we believe that our bio-amplifier can be used to detect leakage of inducible promoters, but because of limited time, we had to focus on our main project. We will complete this research later.
II. The Leakage Coefficient: a value which can represent the level of the leakage of an inducible promoter
Since we've found a method to judge whether an inducible promoter have a leakage or not qualitatively, a value which can represent the level of the leakage of the promoter is needed for quantitative measurements and experiments.
To achieve this aim, we put forward a concept which defines a value called the Leakage Coefficient. The Leakage Coefficient is a value which can represent the level of the leakage of an inducible promoter, and each promoter has its own specific Leakage Coefficient under the same condition.
1. The definition of the Leakage Coefficient
The Leakage Coefficient is a value shows the expression level of an inducible promoter's downstream gene when the promoter is not induced.
2. The measurement and calculation of the Leakage Coefficient
To measure and calculate the Leakage Coefficient of a promoter, a specific system must be used.
Firstly, the measurement system must use our bio-amplifier with a specific standard RBS, BBa_B0034, and a GFP generator, BBa_I13504, must be added after the bio-amplifier.
Secondly, the construction of the measurement system must use iGEM official protocols, and the whole system must be cloned into plasmid pSB1C3.
Thirdly, the protocol of the measurement experiment is similar to the protocol of the InterLab Measurement Study, and a series of devices should be created:
Positive control1: J23100 +B0034 + K2310000 + I13504 in pSB1C3
Positive control2: J23100 + I13504 in pSB1C3
Negative control: I13504 in pSB1C3
Test Device1: The promoter to be measured + B0034 + K2310000 + I13504 in pSB1C3
Test Device2: The promoter to be measured + I13504 in pSB1C3
After preparing these devices, transform them into E.coli DH5α, and measure the absorbance and fluorescence using the protocol of the InterLab Measurement Study. Record the value of FI/ABS600 at the 6th hour of each group, then calculate the Leakage Coefficient with following formula:
LC: Leakage Coefficient
FT1: FI/ABS600 of Test Device1
FT2: FI/ABS600 of Test Device2
FP1: FI/ABS600 of Positive control1
FP2: FI/ABS600 of Positive control2
3. Future work
Just as what we mentioned before, this method to judge and measure inducible promoters was developed accidently, and because of the limited time, we only proved that some inducible promoters assuredly have leakages and our method based on our bio-amplifier can be used to detect it easily, but we have no time to measure more promoters and calculate their Leakage Coefficients, just put forward a concept which defines the Leakage Coefficient. We will try to improve and complete this theory later, and we also decide to measure more promoters, calculating their Leakage Coefficients.
By the way, we want to collaborate with iGEM Foundation to complete and generalize this theory, as well as let more iGEMers and teams participate in this research.
Xiamen University, Fujian, China
No. 422, Siming South Road, Xiamen, Fujian, P. R. China 361005