Team:BIT-China/Collaborations
BIT-China for BNU-China
This year we collaborated with BNU-CHINA team and held a meeting on July 11st, 2015. During the meeting we exchanged our ideas and discussed on our projects. Finally, we confirmed our collaboration relationship.
Because most of BNU-CHINA’s team members were freshmen for iGEM, after the discussion, we decided to help them for parts of their gene circuits. Meanwhile, we had some difficulties in modeling and expressing functional gene NhaA and NhaB, so they offered help to solve these problems.
Fig.1 meeting with BNU-China
We helped them finish the light regulation system (Pcons+B0034+PcyA+B0034+ho1). In this system, Pcons is one of the constitutive promoters (from BBa_J23100 to BBa_J23119) which can be used to tune the expression level of expressing part and what we chose was J23100. B0034 is a kind of strong ribosome binding sites (RBS). PcyA and ho1 are two requisite genes which are required for the transformation from heme into phycocyanobilin (PCB). Ho1 will oxidize the heme group and then generate biliverdin IX alpha, and PcyA converts biliverdin IX alpha into PCB.
Fig.2 Gene circuits of light regulation system
Overlap extension PCR (OE-PCR) was used to finish their gene circuits. However, there were secondary structures in this part. So we could not get the part at first. Then we changed DNA polymerase Pfu to Primer STAR, which performs better than Pfu. Finally the light regulation system was constructed successfully.
Fig.3 The construction result of light regulation system
In this period, we also had some discussions about experiments and modeling face to face. Through these discussions, we learnt more about each other and made progress together.
Fig.4 Discussions with BNU-China
BNU_China for BIT_China
Click here to see BNU-CHINA's description of our collaborations.
○ Modeling
BNU-China mainly helps us to establish a model of substance changes during the pH regulation stage. Specifically, they build a model to link six chemical reaction processes together, all of which are controlled by switch in pH. With change in pH, main chemical reactions will change subsequently. We provided them the conditions and equations of each chemical reaction stage, and they are responsible for the realization of the equations and the whole regulation process.
The difficulty we BIT-China meet is how to connect the six groups of chemical reactions together with the switch in pH, how to identify the reaction time and the results of each chemical reaction, and how to draw an image to reflect the pH variation as function of time. Because some members of BNU-China are majoring in physics, math, computer science and chemistry, they have much experience in differential equations, application of MATLAB and designing computer programs. With these resources, they have helped us solve our problems successfully.
The description of variables are written as followed:
Table 1. The description of variables used in differential reaction equations
Meanwhile, differential reaction equations of the chemical reactions are recorded.[Expand]
After several times of discussion with us, BNU-China understood the background of our modeling part and the problems they were about to settle. Meanwhile, they helped us to find some bugs in modeling, such as the inconsistency of some variable values in each group of equations, and the problem that parameters of some equations don’t accord with the fact.
First of all, BNU-China adjusted a series of independent variables and corrected the parameters. Then they found the solution of the equations by applying the Runge-Kutta Method. The method could be used in differential equation system in different dimensions with high efficiency. According to concentration of hydroxyl ion in equation ABCDE groups, pH of each equation was obtained and was used to judge the termination condition. In equation C and equation F, concentration of specific substances were used to stop the conditions. The initial value of each equation group was the terminal value of the last equation group. By this way they achieved the whole cyclic process and finally got dynamic image of pH(Fig.5).
Fig. 5 The dynamic change of pH during the regulation process
The initial concentration of OH- is 0.2×10-6, and the initial pH is higher than 7.0, so the whole reaction starts from Group A, and the reactions are in the order of Group A, B, C, D, E, F, A, B, C, D, E, F.
○ The detection of protein NhaA and NhaB by BNU_China.
BIT-China’s project this year is to regulate the pH in fermentation environment and to strengthen the competence of microorganism to survive in both acid and alkali. Therefore we want to express two kinds of membrane proteins, NhaA and NhaB. Considering the fact that membrane proteins are particularly hard to express and detect, BNU_China helped us to establish this part. To this end, they reviewed amounts of relevant articles and consulted several professors in this field, on which basis they tried various protocols to express, purify and detect NhaA as well as NhaB.
Initially BNU-China did the SDS-PAGE of the whole protein of bacteria, and the result was not as expected. The first time of result is not shown here. To put it in detail, they centrifuged 5 mL bacterium solution under overnight shake culture at 4 °C, 6000 rpm for 10 minutes, which was boiled for 10 minutes, then protein loading buffer was added. Afterwards they did the SDS-PAGE of the mixture with the running gel concentration 12%.
Because the whole cell was too stick, BNU-China couldn’t make an accurate estimation from the result, but anyway there was no sign of expression of the objective proteins whose molecular weight was about 46 kDa.
Then they tried a normal SDS-PAGE of the homogenate and supernatant and the result is shown in Fig.6
Fig.6 Normal SDS-PAGE of NhaA and NhaB
Lane 1, molecular weight standards (kDa); Lane 2-4, the homogenate of pSB1C3, NhaA and NhaB; Lane 5, 6 and 8, the supernatant of pSB1C3, NhaA and NhaB, Bacteria transformed with plasmid pSB1C3 as a negative control.
The bands were clearer this time, but there was no significant difference between the empty vector plasmid and the transformed ones.
After two times of unsuccessful attempts, BNU-China reviewed some articles of reliable protocols for membrane extraction and detection. With the protocols collected, they processed the experiment the third time(Fig.7).
Fig.7 SDS-PAGE of extracted membrane proteins
Lane 1, molecular weight standards (kDa); lane 2-4, the sediment of pSB1C3, NhaA and NhaB after centrifuging for the second time; Lane 5-7, the supernatant of pSB1C3, NhaA and NhaB after centrifuging for the second time; Lane 8-10, the supernatant of pSB1C3, NhaA and NhaB after centrifuging for the first time. Bacteria transformed with plasmid pSB1C3 as a negative control. From this result BNU-China concluded that the differences exist between the bands of controlled plasmid and the experimental ones, whereas it seemed that these differences had nothing to do with the objective product. Hence, it came out as another failure.
Relevant agent formula:
protein lysis buffer
Tris-HCl (pH=8.0) 85 mL
Sucrose 25.67 g
β-mercaptoethanol 15 mL
membrane solution buffer
protein lysis buffer : urea solution (8M) = 4 : 1 (volume ratio ))
Therefore, it was a pity that BNU_China failed to detect those two proteins, but they were able to give some opinions to us about protein detection through their experience:
1. Changing the expression strain for overexpressing of NhaA and NhaB according to the citation [1], the details are in Table. 2.
2. Make western blots if possible.
The experiments gave us lots of information about membrane proteins. What’s more, the experience helps us two teams communicate frequently and therefore develop a tight relationship eventually.
Table. 2 Bacteria strains specially for overexpressing NhaA
BIT_China for TJU
TJU is our old friend in the iGEM stage; we had collaborated with them in experiment, modeling in past two years.
P170 is a kind of acid-induced promoter, and can be bound by rcfB protein activated by lactic acid. To verify the relationship between concentration of lactic acid and promoter, P170 was linked with RFP gene, and strength of fluorescence was measured. Fig.8 shows the circuit with promoter P170.
Fig.8 Genetic regulation circuit of promoter P170
We firstly made assumption of the circuit:
(1) mRNA and combination of mRNA-rib had reached steady state
(2) all enzymes activities were not affected by acid concentration and could be regarded as constant
(3) concentration of rcfB protein was assumed unchanged.
The reaction processes are described as:
Here we list the parameter names and descriptions:
Basing on reaction processes above, equations are constructed as followed: Concentration of induced RcfB and combination of RcfB and lactic acid are written as:
Where KLa and K are constants and n is hill coefficient.
Promoter P170 can be activated in two ways. The first pathway is to bind to the dimerization of lactic acid and RcfB. The other pathway basing on our assumption is that a proportion of P170 can be activated directly.
Thus, the concentration of activated P170 promoter is described as:
Where km1 and km2 are integration parameters. As transcription and translation processes could enter steady state in a short period of time, here we directly considered steady state of mRNA, mRNA-rib and RFP, where
Finally, we calculated fluorescence:
The equation demonstrated the connection between fluorescence and lactic acid, thus, the concentration of lactic acid could be measured by fluorescence through the system. We set km1=10 and use promoter J23100, then results showed how these different substances changed with the lactic acid concentration. (Fig. 9)
Fig.9 Concentration of P170, mRNA, mRNA-rib and fluorescence change with the lactic acid.
Fine tuning:
As RcfB plays a key role in induction of P170 promoter, we firstly conducted constitutive promoter fine-tuning. Different constitutive promoters were constructed with downstream RcfB. We built up models to compare the function of these promoters (Fig.10). Results showed that J23100 had the highest effectiveness.
Fig.10 Comparison of fluorescence led by different constitutive promoters.
Parameter table:
TJU for BIT-China
Click here to see TJU's description of our collaborations
Basic circuits contain two subsystems: resistance subsystem and regulation subsystem. Models of these two subsystems were constructed separately.
Main processes include induction of the promoters, transcription and translation of function genes, catalysis of enzymes and acid or alkali production led by enzymes. ODE is used for modeling to describe concentration as function of time. Factors considered are production and degradation, combination and disaggregation and reversible reaction.
Resistance subsystem consists of constitutive promoter J23119, function genes glsA, NhaA and NhaB. J23119 is not influenced by environmental pH, and will lead enzymes production continuously.
Fig. 1 Basic resistance device.
Our model mainly focused on functional genes and enzymes. Assumptions we made are:
(1) enzymes will only work in proper pH range and are not activated at other pH level.
(2) To prove function of our device, initial intracellular pH is set to ideal value.
For acid resistance device (Fig. 1), description of reaction processes are:
Where description and initial value of variables are :
Fig. 2 Name, initial value and description of variables.
Finally, result showed that with application of acid resistance device, pH could be regulated to a suitable range (Fig. 3).
Fig. 3 Intracellular pH is regulated by acid resistance device. To prove the function of circuit, initial pH was set up to 3.0, when glsA was activated. pH can effect the activity of glsA, while glsA conducts gaseous ammonia production. With the enzyme activity changing, pH will be finally stabilized at a suitable level.
For alkali resistance device (Fig.1), NhaB and NhaA genes’ functions were assumed as similar processes to enzyme catalysis. Basing on this assumption, description of the NhaB circuit is:
Consistent with equations above, functions are:
Where description and initial value of variables are:
Fig.4 Name, initial value and description of variables.
As for NhaA, the reaction processes are:
And the functions are:
Where description and initial value of variables are:
Fig.5 Name, initial value and description of variables.
As the result, alkali resistance device could decrease intracellular pH (Fig.6.1, 6.2).
Figure.6.1 Intracellular pH change regulated by alkali resistance circuit led by NhaB. NhaB is not effected by pH, and can assist NhaA to regulate alkaline environment. Here we assumed that initial pH was 9.0 and NhaB had already been activated.
Figure.6.2 Intracellular pH change regulated by alkali resistance circuit led by NhaA. NhaA will be induced at pH 7.0 ~ 9.0, while NhaB will function when NhaA cannot function normally. Here we assumed that initial pH was 8.0 and NhaA had already been activated.