Production Module

Goal

    1. To express exogenous SAMe synthetase opSam2 and SAMe transporter opPet8p in Bacillus subtilis.
    2. To prove that opSam2 can synthesize S-adenosyl methionine (SAMe) and that opPet8p can transport SAMe, respectively in Bacillus subtilis. .

Achievement

    1. The expression shuttle vectors of opSam2 and opPet8p were successfully constructed and transferred into Escherichia coli (E. coli) DH5α and Bacillus subtilis (B. subtilis) 168.
    2. Successful Expression of opSam2 and opPet8p in both E. coli DH5α and B. subtilis 168.
    3. Successful membrane localization verification of opPet8p in B. subtilis 168
    4. Functional verification of opSam2 in B. subtilis 168.

Experiment

Construction of expression vector of pHT-opSam2 and transformation

    In order to ensure that our exogenous protein opSam2 can be expressed in gram-positive B. subtilis 168, and deeper in Bifidobacterium longum (B. longum), we used a E. coli - B. subtilis shuttle vector pHT01K (8253bp, with Kan resistance), which is derived from original pHT01 (with Cm resistance). This plasmid gained from our PI Xie’s lab, has an intrinsic lactose-inducible promoter Pgrac, and this plasmid can express Pgrac repressor LacI, so the expression of protein of interest inserted can be induced by IPTG, a lactose analogue. We inserted our synthetic fragment 6xHis::opSam2 into MCS of pHT01K, using enzyme cut and ligation to construct the recombined plasmid pHT-opSam2 (9506bp). After transformation, we performed colony PCR and extracted plasmid to run agarose electrophoresis. The correct band was observed (Fig. 1.1A, B, C and D), and the sequencing results verified the successful insertion in B. subtilis as well (Fig. 1.1E).

Construction of expression vector of pBE-XylR-opPet8p and transformation

    In order to ensure that our exogenous protein opPet8p can be expressed in gram-positive B. subtilis 168, and deeper in Bifidobacterium longum (B. longum), we used a E. coli - B. subtilis shuttle vector pBE2 (6292bp), which also makes co-transformation with pHT-opSam2 available. This plasmid was gained from our PI’s lab as well. It has merely a common prokaryotic S6 promoter, which can’t satisfy our need to verify the inducible expression. So we firstly inserted a P43-promoted XylR fragment into the plasmid between PstI/KpnI double cut sites, and then we inserted another fragment Pxyl-promoted opPet8p into the first recombined plasmid between KpnI/EcoRI double cut sites. The XylR fragment was amplified through PCR from genome of B. subtilis, and the other fragment was synthesized. Similar to the procedure done in pHT-Sam2, pBE-XylR-opPet8p (10046bp) has been constructed and transferred into E. coli and B. subtilis (Fig. 1.2).

Expression of opSam2

    In order to verify opSam2 expression, we utilized the 6xHis tag fused to N-terminal of opSam2 to perform Western Blot. Since the opSam2 is downstream of Pgrac, a lactose-induced promoter, we used IPTG to induce its expression in E. coli first.
    To enlarge the expression of opSam2, we choose the induction condition at 1mM IPTG and 37˚c according to the literature. Then, we prepared loading samples at different time after induction. We firstly ran SDS-PAGE and used Coomassie staining to verify the lysis of bacteria and protein input (Fig. 1.3A). Then to compare the induction efficiency and detect opSam2 expression, we used BCA quantification method to measure the concentration of total protein (Fig. 1.3B).
    Then after the concentration measurement, we diluted samples into almost the same concentration, and used such diluted solution to perform western blot using antibody against His tag (Fig. 1.3C and D). In this case, we can correspond the band brightness of protein of interest to the concentration directly.

    As the result shows, we can figure out that the band between 40 and 55 displays the existence of 6xHis::opSam2 whose molecular weight is predicted to reach about 45.80 kDa. As the induction during increased, the band brightness became darker, which means that the expression of 6xHis::opSam2 is increased. Meanwhile, we may find some shifting phenomenon in right several lanes like lane 6, 7 and 8. This could be caused by the degradation of opSam2 due to long time of induction and lysis. So when we performed WB of opSam2 in B. subtilis, we strickly controled the lysis time and reduce the induction time. Thus, the result looks clearer and more specific (Fig. 1.3D). The target band obviously became darker when induction time increased. So we can conclude that the expression of opSam2 in B. subtilis succeeded.

Expression of opPet8p

    In order to verify opPet8p expression, we utilized the 6xHis tag fused to N-terminal of opPet8p and the sfGFP tag fused to C-terminus to perform Western Blot. Since the opPet8p is downstream of Pxyl, a xylose induced promoter, we used xylose to activate its expression.
    We synthesized the fragment Pxyl-6xHis::Mistic::opPet8p::sfGFP from Tsingke Biology. We found that without XylR, the repressor of Pxyl, bacteria containing such fragment can constitutively express 6xHis::Mistic::opPet8p::sfGFP, for we observed the green fluorescence in colonies in the plate when they were excited by UV light (Fig. 1.4A) and turned green under visual light. However, when inserted together with P43::XylR, bacteria cannot obviously emit green fluorescence visible to the naked eyes, so we concluded that the Pxyl could work and XylR could repress Pxyl as reported.
    To induce the expression of opPet8p in E. coli and B. subtilis, we tried 0.3%, 0.8% and 1% xylose to induce, and finally we used 1% xylose to induce since its efficiency is best. When preparing the protein sample after xylose induction, we can easily figure out the expression of opPet8p::sfGFP in E.coli and B. subtilis through sfGFP tag. Only the image in E. coli was taken (Fig. 1.4B). Meanwhile, we performed fluorescent microscopy of E. coli and B. subtilis with pBE-XylR-opPet8p after induction (Fig. 1.4C). The results shows the successful expression of opPet8p::sfGFP. After induction and lysis, we performed BCA quantification (Fig. 1.4D) and dilution to make sure that the loading mass of total proteins remained the same, then we performed western blot to detect opPet8p expression level in both E. coli and B. subtilis (Fig. 1.4G). We used two kinds of antibodies, one against sfGFP (Fig 1.4E) and another against 6xHis (Fig 1.4F).

    We can figure out that as the induction during increased, the expression of His-conjugated protein increased. Meanwhile, the induction or expression efficiency in B. subtilis is higher than in E. coli, according to the optical density of the lanes. It’s easy to understand, the promoter Pxyl is endogenous for B. subtilis and it will be more suitable to be activated in it than in E. coli. However, what confused us is that the molecular weight observed (about 30kD) is different from what we predicted (73kDa). We thought it could be some particular mechanism to cut such protein during maturation so that it became smaller, but the transcription and translation are working normally since both the N-terminal signal His tag and the C-terminal sfGFP can be detected.
    For the above reason, we used another antibody anti-GFP to perform Western Blot (Fig. 1.4G), and the results shown that there are several truncated proteins except full-length protein (73kD) (Fig. 1.4E). So further experiments should be done to learn about the existing state of opPet8p in B. subtilis. According to above results, we can conclude that B. subtilis can expressed the recombined protein opPet8p, while the precursor proteins might be cleavaged to mature proteins.

Membrane localization of opPet8p

     Since the opPet8p is membrane-bound, we tended to find out whether it can localize in membrane of prokaryotic cells. To better understand the membrane integration of our recombined protein opMistic::opPet8p::sfGFP, we used a prediction tool Phobius to predict . The results are showed below (Fig. 1.5A). As we can see, the addition of Mistic can result in the addition of transmembrane segment which can facilitate the membrane insertion of opPet8p, while the sfGFP does the opposite thing. Thus after using sfGFP to verify its expression and membrane localization, if we want to improve its membrane localization efficiency, we need to replace the sfGFP with other tag, like some small particular fluorescent group. Then we used xylose to induce the expression of opPet8p-sfGFP in B. subtilis, and then take some bacterial solution to perform fluorescent microscopy (Fig. 1.5B). The group constitutively expressing opPet8p without repressor XylR is on the right. In groups with XylR, the expression is induced by xylose. Treated with different concentration of xylose (Fig. 1.5B Left with 0.5% and Middle with 1%), we can briefly observe the density of fluorescence became stronger when bacteria were induced with higher concentration of xylose. However, the particle-like distribution became more obvious when bacteria were treated with more xylose.
    So according to the distribution of fluorescent signal, we supposed that the recombined proteins might target to the bacterial membrane. However, if the expression rate is too high to conduct the proper folding so that they might form the particle-like inclusion body, they might gather together to precipitate.

Catalytic ability of opSam2

    Since we have verified the expression of SAMe synthetase opSam2, we next measured its catalytic capacity of synthesizing SAMe from ATP and methionine. We induced opSam2 expression in B. subtilis, then aspirated the supernatant, and took the pellet to measure the SAMe concentration through SAMe quantification ELISA kit (Fig. 1.6A). The standard curve demonstrated our result was credible. In this experiment, we set a control group with empty vector (EV), which did not contain the recombined protein gene but contained the primary plasmid pHT01K, so we can use this group to standardize the concentration change of experiment group (Fig. 1.6C and D).

∆Relative concentration = (Induced Experiment Goup – Induced Control Group) / (non-induced Experiment Group – non-induced Control Group)

We can figure out that the after induction, the concentration of SAMe in Experiment grouo (pHT-opSam2) and Control (Empty Vector) group both definitely increased. But the increase of Experiment ground is sharper than control group. Besides when we used the control to standardize the concentration change of Experiment Group, we found a obvious increase of relative concentration change in Experiment Groups. we can conclude that opSam2 can function normally in gram-positive bacteria B. subtilis, which means that it would be greatly possible to work in our final target probiotic Bifidobacterium longum.

Oscillator Module

Goal

    1.To express lacI, tetR, lambda CI in B. subtilis, and verify their function, as in if they can repress the expression starting from their corresponding promoter.
    2.To test the function of the plasmid Repressilator, in E. coli.

Experiment

Expression of lacI, tetR, lambda CI

     In order to construct a plasmid that contains both the transcription factors and a GFP whose expression is driven by the corresponding promoter, we chose to use a shuttle vector, pBE2, as the chassis vector to construct our plasmid, for its convenient ability to be able to replicate in E. coli that allows us to finish most of our experiments without using B. subtilis, to ensure higher possibility of success.
    First of all, we decided to verify the function of the constitutive promoter, P43, that we chose as the promoter that would drive the expression of the transcription factors (e.g. lacI), so, we first designed the plasmid that used P43 to drive the expression of GFP.
     We obtained the plasmid backbone of pBE2 from Zhixiong Xie Lab. GFP sequence was obtained from part BBa_E0840 that was given by iGEM foundation, and P43 sequence was synthesized by Tsingke Biotechnology company. After obtaining the sequences, we designed primers to amplify the desired part of the sequence, and after gel electrophoresis, band of the expected size was observed.

Figure 2.1 M: Marker; 1-2: PCR product of plasmid; 3-4: Unsuccessful attempts to amplify the Vector by PCR; 5-6: PCR product of GFP fragment; 7-8: PCR product of P43 promoter; 9-10: Unsuccessful attempts to amplify the promoter by PCR; 11-12: PCR product of P43 promoter

But after we used gel purification to purify the corresponding band, the concentration of the vector was quite low, so, we performed another round of PCR, and obtained linearized vector with sufficient concentration.

Figure 2.2
    Left: M: Marker; 1: Unsuccessful attempts to PCR amplify the vector; 2: Unsuccessful attempts to PCR amplify the vector; 3: PCR amplification of the vector
    Right: M: Marker; 1-4: PCR amplification of the vector

We performed Gibson assembly, and transformed the ligation product into DH5α competent cells. And after incubating the plate for 20h, we used colony PCR to verify the positive transformants. The result is shown below. (We didn’t manage to culture the bacteria after the first round of colony PCR, so, we did another one.)

Figure 2.3 Left: First round of colony PCR. Right: Second round of colony PCR. M: Marker; +: Positive results; -: Negative results

After selecting out the positive transformants and culturing them, we extracted plasmid, but due to the lack of time, we didn’t manage to get to the part of functional verification.

Test the function of the Repressilator plasmid in E. coli

    Other than verifying the three transcription factors one by one, we also planned on testing the function of Repressilator as a whole. So, we decided to transform the Repressilator plasmid and a reporter plasmid that consists of GFP whose expression is driven by tetR promoter.
    The Repressilator plasmid was ordered directly from Addgene, and the reporter plasmid we chose was obtained from iGEM kit.
    After co-transformation, we used double-antibiotic plate selection and colony PCR to pick out the transformants that had both plasmids (Fig 2.4). And we successful gained co-transformed strain.

Figure 2.4 M: Marker; P.C.: Positive Control

    To test the fluorescence of the co-transformed bacteria, we incubated the bacteria overnight, diluted the culture for 100 times, and incubated the diluted culture for another 2 hours. Then, we dropped a drop of molten solid LB media on top of a glass slide, dropped another drop of bacteria culture, spread it evenly, and observed under the fluorescence microscopy. We did observe fluorescence, but we didn’t observe the fluctuation of it. We hypothesized that this was due to the fact that the oscillation cycle was too long, and in fear of the quenching of GFP molecule or adverse effects exerted on our bacteria due to long time exposure of blue excitation light, this oscillation cycle can’t be observed continuously under the microscope, so, we decided to take photos of the same area of the slide with the same setting and at given time intervals. We settled on a time interval of 10 minutes, for a whole duration of 1 hours, and with support from math models, we managed to solve the problem of the measurement of the oscillation cycle of the Repressilator. The solving process is shown below.

Figure 2.5 The original photo of the Repressilator taken at an time interval of 10 minutes.

    Then, we measured the light intensity of the whole photo, to serve as the background of the fluorescence intensity (making a hypothesis that the light intensity of the bacteria with fluorescence is so much less than the background that the light intensity of the bacteria can be neglected, and as can be seen below, this hypothesis is correct)

Figure 2.6 The measurement of the light intensity of the whole photo, to see the background.

    The first box indicates the average light intensity per pixel of the whole photo, and the second box indicate the number of pixels of the image. The whole light intensity can be obtained by multiplying the two numbers together.

    Then, we cycled out the same bacteria of each photo, making sure that they are all at the same region in each photograph, numbered them 1-10, as shown in figure a, and measured the light intensity in each of the small regions, the result is represented in figure b, and the data obtained is shown below in the table.

Figure 2.7 The numbering of each bacterium, and the measurement of the light intensity in regions around each bacterium.

Total number of pixels around each bacterium and the whole picture

Average light intensity around each bacterium and the whole picture

Total light intensity around each bacterium and the whole picture without background

Figure 2.8 Analysis workflow of light intensity around each bacterium and the whole picture.

    To visualize these data, we imported them into Prism, and performed non-linear regression using sine waves as the model, to find out the expected oscillation cycle of the Repressilator plasmid. The result is shown below.

But as can be felt from the regression curve, the data point at 30 minutes violated the whole trend, and the goodness of fit is quite low. So, we decided to delete this data point, and do the regression once more.

And as can be seen, the result is much better, and the oscillation cycle settled around 60 minutes, which agrees with the result shown in the original article.

Directed Evolution Module

Goal

     1. Construct a plasmid containing reporter genes for further selection.
    2. Build mutagenesis library of lacI.
    3. Separate desired LacI protein that can tolerate relatively high level of lactose.

Achievement

1.The plasmid is successfully constructed and site-saturation mutagenesis is successfully introduced;
2.The mutated plasmids are transformed into E. coli BL21(DE3) and successfully build up the mutagenesis library.
3.The desired LacI protein is successfully derived through rounds of selection process.

Experiment

Plasmid construction

    Our selection plasmid intends to contain three selection elements: mRFP1, sacB and KanR (Fig 3.1A). In the beginning, we planned to use overlap extension PCR to connect each fragment into one and to combine it with linearized vector (BBa_J04450) as one composite part. However, the fragment was quite unstable and was easy to break even once successfully connected (Fig 3.1C). Thus, we decided to directly use Gibson Assembly for the whole construction by separating the process into building two intermediate plasmids (Fig. 3.1B) with only two to three fragments at a time. Even though the success percentage wasn’t 100%, as fragment loss still occasionally occurred, we successfully constructed the final plasmid using this principle (Fig. 3.1D).

Figure 3.1 Plasmid construction and Electrophoresis analysis.
A: Diagram of the final plasmid.
B: The first intermediate of plasmid construction.
C: Overlap extension PCR product of sacB and KanR break up at the next amplification.
D: Colony PCR of random transformant, identifying three fragments respectively.

We also optimized the element as we realized the possibility of lacI mis-expression in previous design because of the inappropriate distance between RBS and the stating codon of lacI , by deleting some base pairs between RBS and lacI gene (Fig 3.2).

Figure 3.2 Sequencing of developed lacI expression section with expected deletion occurred.

Building variant library

    We decided to use MEGAWHOP (The Megaprimer PCR of Whole Plasmids) to introduce mutagenesis into LacI gene. To construct megaprimers with site-saturated mutagenesis, we used overlap extension PCR. After DpnI digestion, we transform the variants into E. coli BL21(DE3) and obtain the mutagenesis libraries on on chloramphenicol-added plate (Fig 3.4a). The colonies were selected for sequencing, expected mutations were observed at target positions (Fig. 3.4).

Figure 3.3 Mutagenesis library construction.
A: No megaprimer in MEGAWHOP;
B: Megaprimer added in MEGAWHOP. DpnI digestion is applied to both group, and non-mutated plasmid can’t be transformed into competent cells.

Selection

     Based on sacB and KanR added to our plasmid, our selection can be delivered by selective culture of mutants. Since sacB allows negative selections on mutant libraries, details in Design, we first carried out experiments with sucrose medium, exploring multiple conditions to execute the selection including sucrose content and induction duration etc. However, we failed to separate the ideal mutants in this way. Later test suggests that the reason may be the wrong timing for induction or spreading, as the killing effect being significantly distinguishing when we spread the culture on medium with 8% sucrose and 0.8mM IPTG at OD600 equal around 0.2. For detailed information please check on Engineering.
    Due to the time constraint, we turned to establish comparison experiments using KanR as report gene. Gradient IPTG were added into 50 ug/ml kanamycin solid plate to observe variant’s performance in different induction concentration. Our results also once again showed our success in mutagenesis library construction, with various kinds of behavior occurred in different mutant strains (Fig 3.4 C-J).

Figure 3.4 Partial results of selection.
A: Control group 1. Original strain and random mutant are applied on empty antibiotic plate with IPTG contrast.
B: Control group 2. Original strain is applied on Kan+ plate with IPTG gradient.
C: Desired mutant, Strain #29. No colony growth is observed at 0 and 0.1mM IPTG, suggesting that the LacI can tolerate high-level IPTG.
D: Strain #27. The mutagenesis causes its binding to lactose (IPTG) to be easier, and lawn are witnessed at all concentration, even without IPTG existence. Its behavior is quite familiar to performance on non-antibiotic medium.
E: Strain #9. The mutagenesis slightly reduces the leakage of the promoter, however, barely difference occurred between different inductor (IPTG) concentration.
F: Strain #24. Little up-regulation of the promoter takes place as IPTG concentration lifting. Its response to low-level inductor (IPTG) is still relatively high.
G: Strain #2. Few colonies occur at 0mM IPTG and no growth at higher level.
H: Strain #6. No growth at all. The sensation to IPTG is totally lost.
I: Strain #13. Expression is activated only at 0.1mM IPTG.
J: Strain #14. Few colonies are witnessed, expect one at 0.5mM IPTG, suggesting its affinity to IPTG has greatly dropped.

    In order to evaluate the behaviors of different mutants for better judgement, we designed a algorithm to access the IPTG responsive ability of the strains. We used number 1-7 to measure the intensity of the colonies on each plate, and integrated them into a heatmap for visualization (Fig 3.5). Our goal is to select a LacI variant that cannot sense the low concentration IPTG and at the same time normally functions at with concentrated IPTG induction. Thus, reflecting on different colors, the ideal strain are supposed to have lighter color between darker ones at high range of IPTG. Comparing the data, Strain #29 is exactly the one that we are looking for (Fig 3.4 C). Meanwhile, a few growths under 0.8mM IPTG guarantees that the mutant LacI still keeps the ability to release from promoter and it could function normally in our oscillatior - “Repressilator”. Selected mutant also appeared to solve the leakage problem that original LacI contain, showing a more outstanding performance.

Figure 3.5 Visualization of arithmetic LacI evaluation result. The triangle points out the desired variant strain #29.

    After sequencing, we are surprising to find that the mutations are taken somewhere else than the positions we set. This situation could happen as our mutagenesis library contains the original sequence, and new mutations may be introduced during multiple amplification reactions during the construction process. The ideal results however suggest that it’s highly possible that these are other residues that may contribute to LacI’s substance binding ability. Further clarifications could be delivered to explore the functions that these residues may hold.
    Limited in time, we weren’t able to measure the characters of this variant. However, present results are convincing enough to indicate the success in our selection. The improved LacI repressor is documented as a new part BBa_K4144041.

Safety Switch Module

Goal

    1. To construct two composite plasmids which work as chemical and temperature switch respectively.
    2. To test the function of these composite parts in B. subtilis.
    3. To test the lytic strength of bacteria when PBSX holin expresses.

Achievement

    1. The temperature switch with EGFP as a reporter can be induced by low temperature.
    2. The expression of PBSX holin has the ability to inhibit the growth of the bacteria to some extent.

Experiment

    In order to test the function of two composite plasmids which work as chemical and temperature switch respectively, we first chose EGFP as their downstream reporter. We have constructed these plasmids successfully (Fig 4.1).

Figure 4.1: The result of plasmid construction and sequencing. A, B: Construction and sequencing of chemical switch. C, D: Construction and sequencing of temperature switch.

    Firstly, we tested the function of the plasmids in E. coli DH5α.
    For chemical switch, we treated engineered bacteria with CinI, a molecule for quorum sensing which can form a complex with CinR to induce expression of downstream EGFP or Holin. We used recombined E. coli BL21 to produce autoinducer and extract the supernatant. We tested the function of chemical switch by inducing the expression of EGFP with the supernatant.

    For temperature switch, in the environment below 36 degrees Celsius, Temp will work to activate the expression of downstream EGFP or Holin, while over 36 degrees Celsius, Temp will be shut down and bacteria can’t emit green fluorescence or be killed. To determine whether Temp can work smoothly, we incubated E. coli containing pBE2-Temp-EGFP overnight and then equally divide bacteria into two EP tubes, cultured respectively at 25 and 37 degrees Celsius for one hour.
    As a result, unfortunately, no fluorescence was observed among different groups of chemical switch. But the fluorescence of temperature switch induced at 25˚c was observed (Fig 4.2A and B).
     After that, we transformed the plasmids into B. subtilis successfully and quantitively measure the expression of fluorescence (Fig 4.2 C and D).

Figure 4.2: The fluorescence in E. coli with chemical or temperature switch and the fluorescence in B. subtilis with chemical or temperature switch.
     A: Chemical switch in E. coli
     B: Temperature switch in E. coli: The short rod-shaped E. coli cultured at 25 degrees Celsius fluoresces green, while the bacteria cultured at 37 degrees do not fluoresce. This indicates that Temp induced by low temperature can initiate the expression of downstream genes, while Temp does not work at high temperatures. The results are in line with our expectations.
     C: Chemical or temperature switch in B. subtilis:
1) Relative to the control group, values of chemical-induced+ inducer increased to some extent, but the margin wasn’t significant enough.
2) Chart shows data in the second hour. Values at 37 degrees are benchmarks. There was no significant increase in the data of the first two groups relative to the latter two groups, which means EGFP in engineered B. subtilis didn’t express.

    Lastly, we tested the function of PBSX holin by measuring the lysis of E. coli. In this construction, we transformed pET-Holin into E. coli BL21 and the expression of holin is induced by IPTG. We found that after inducing, the bacteria were killed or inhibited to some extent though not fully (Fig 4.3). To qualitatively verify holin’s function, we coated the solid medium with 100mL of E. coli solution and affixed round paper soaked in water on one side, while the other side was affixed with round paper soaked in 1mM IPTG. The plates were incubated at 37℃ overnight and observed for the formation of bacteriostatic circles.

Figure 4.3: Inducing the expression of PBSX holin in E. coli BL21.
    A. Induction on culture plate: After incubation overnight, the bacteria had grown into a moss on the medium. A large number of bacteria still grew around the water-soaked paper, while the growth of bacteria near the paper soaked with IPTG was inhibited, with the number of bacteria decreasing and single colonies appearing.
    B. Change in turbidity: It can be seen that the bacterial concentration of the group induced by IPTG was significantly reduced compared with that of the control group, but there was no significant difference between different induced concentrations. The lytic efficiency did not increase obviously with the extension of culture time. The results showed that holin could work in E. coli.

    Thus, we still need to improve our designs in the future to make them complete safety switches. Though we have proved that the most basic parts in our design will be able to work in B. subtilis, further examination of each single part is still needed. For example, TlpA36 promoter should be tested in B. subtilis to verify whether it has its normal function. What is more, we can use directed evolution method to improve those parts for the expression in B. subtilis.