In our construct design, we eliminated the C-terminus 16 a.a, which can increase the efficiency of SHM while inhibiting the function of CSR (see Design). However, the NES (nuclear export signal) overlapped the CSR. Because the AID translated into the cytoplasm must diffuse into the nucleus to function, we were concerned that the elimination of NES would change the diffusion patterns of the AID or even have a negative effect. Therefore, we decided to build a model for dNES AID. We described the kinetic process of nuclear transport of the AID and other transport factors with ordinary differential equations to build a model. With this model, we qualified the nuclear-cytoplasm exchange of the wild AID and the NES-deleted AID. More details are described on the Model page.

Before testing, we built a model describing the concentration of the expressed AID. Then, we incorporated this result into the NES model. After calculating by MATLAB, we got the following result (Fig. 1). The blue line represents the diffusion of the wild AID, while the yellow line represents that of the NES-deleted AID. In addition, the result also shows the concentration change of nuclear transport factors, such as importing, exportin-RanGTP complex, etc. More details are described on the Model page.

The yellow line almost overlaps the blue line (Fig 1.a), which means that the deletion of the NES has no effect on the transport of AID and doesn’t change the concentration of AID in the nucleus (Fig 1.b). The results(See Model) gave us evidence that we could eliminate the C-terminus 16 a.a, inhibiting the function of CSR without affecting the nuclear diffusion of the AID. Therefore, we decided to continue using the construct of C-terminus eliminated AID.

We searched for all protocols to maintain the hybridoma offered by ERABIOTEQ ENTERPRISE CO., LTD, in good condition. After studying several researches, we found that DMEM and RPMI might be two of the best culture medium candidate for our hydridoma cell.

After testing RPMI-1640 and DMEM on our hybridoma, we discovered that the hybridoma cell’s death rate in RPMI is lower than in DMEM. However, the hybridoma cell did not grow as fast as we expected in RPMI-1640.

Medium	Cell mortality rate
RPMI	14%
DMEM	27%

After consulting professor, Mr. Jan, research studying[1] and modeling(see Model), we added some growth factors into our medium, such as IL-6 and glucose, to trigger the growth of B lymphocytes. During several rounds of discussion and testing, we eventually came up with our protocol of medium that provided the hybridoma with the best living condition. The concentration of each component is shown in the chart below.

Medium	Cell mortality rate
RPMI	14%
DMEM	27%
RPMI+IL-6	5%
DMEM+IL-6	23%

To examine whether our target gene was successfully inserted into the genome of hybridoma cells and adequately expressed, GFP was chosen as our reporter gene. It can be observed easily under the fluorescence microscope.

Before the transfection of the lentivirus, which carries our target protein, we designed lenti-GFP, a lentivirus that carries only GFP, to examine whether our protocol of lentivirus production and infection can function. The fluorescence microscope result shows that the lentivirus production and infection protocol works since hybridoma expresses GFP adequately. However, we encountered another technical problem. The survival rate of the cell were low. To tackle this problem, we searched for possible causes and found research[2] suggested that GFP might be toxic to immune cells and lead to the malfunction of specific cell lines or even death. Since hybridoma originates from immune cells, we suggested that the expression of GFP might result in a high cell mortality rate.

We discussed with IGEM NCHU and eventually determined that RFP might be a better reporter gene for immune cells. We constructed a lenti-RFP that carries only promoter and RFP gene to test whether it is helpful for cell viability. The graph below shows that using RFP as a reporter gene increase the cell survival rate and might be a better reporter gene for immune cell.

According to former research[3], the higher the concentration of doxycycline, the faster the down stream sequences are expressed. However, high concentration of doxycycline may lead to cellular toxicity and interrupt with the proliferation of cell.[4] To tackle with this issue, we performed a modeling experiment to examine what is the best concentration for Tet-On system to fuction while did not disrupt the growth of cell.

As shown in figure 6 of result from AID-expression model(See Model), we demonstrated that the amount of protein produced by Tet-On system under different concentration of doxycycline(10X serial dilution). The result shows that when the concentration of doxycycline exceed 10μg/ml, the amount of protein expressed is nearly identical. According to this fiding, we tested 0.1μg/ml, 1μg/ml, 10μg/ml of doxycycline in medium and conducted western blot to check the level of protein expression.

As the model of doxycycline-inducible model shows, the expression of protein under the concentration of doxycycline over 10μg/ml is nearly the same. In comparison, the result of the western blot shows that a medium with 10μg/ml of doxycycline has the best effectiveness of protein production. According to the modeling data and the western blot result, we eventually decided to use 10μg/ml as the concentration of doxycycline to induce the Tet-On system.