Result

Prediction of Protein Structure

In order to realize the functions we designed in our project (1. The combination of AFB1 and AFB1-ScFv.2.The combination between Sh3 ligand and SH3 domain.)We firstly need to complete the construction of new biobricks. To realize its functions , our AFB1-ScFv coding sequence must have the features shown below:

1. It can express SH3 ligand which is exposed to the outer surface of the protein. so that it can combine with SH3 domain on the membrane protein we constructed. It can express 6*his-tag which is exposed to the outer surface of the antibody, so that it can be combined with the anti-his antibody. SH3 domain-LGT coding sequence must have the features shown below:
2. It can express SH3 domain which is exposed in the outer membrane, so that it can be combined with SH3 ligand on the membrane protein we constructed in the project. In order to verify our design of protein structure. We put our protein sequence into the I-TASSER online analyzing. http://zhanglab.ccmb.med.umich.edu/I-TASSER

The result is analyzed below.

fig.1: The Prediction Structure of the Fusion Protein.(A) The schematic layout of AFB1-ScFv. (B) The simulated diagram of AFB1-ScFv. The blue part is SH3 ligand. The pink part is 6＊his-tag. The red part is the signal peptide. The white part is the linker between VH and VL. The green part is VL and the orangepart is VH. (C) The schematic layout of SH3 domain-LGT. (D) The simulated diagram of SH3 domain-LGT. The red part is SH3 domain and the orange part is the signal peptide.

As shown in figure 1(B), the 6＊histag and the SH3 ligand is successfully exposed to the outer surface of the protein. And the SH3 ligand can bind with SH3 domain, and the 6＊his-tag can be combined by anti-his antybody.

As shown in the figure 1(D) the SH3 domain is successfully exposed to the outer surface of the protein. And the SH3 ligand can bind with SH3 domain freely. We speculate from all the analysis above that our design of the protein structure is practicable.

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Acquisition of BioBricks

fig.2: The Construction of Biobricks:(A) The construction of biobrick (AFB1-ScFv), which is standardized by the specific upstream and downstream primer. (B) The construction of biobrick(SH3 domain-LGT). The upstream and the downstream of BBa_K771107 and BBa_K771102 were designed with specific primers respectively. The DNA fragment of ssDsbA（signal peptide）and linker+LGT was first obtained through primer PCR. Then the construction was finished by biobrick assembly. (C) The agarose gel electrophoresis of the biobricks in the project. The first lane is DNA marker（the length of the stripes is shown in the figure). The second to the fourth lane are AFB1-ScFv，AFB1-ScFv（nonsp），SH3 domain-LGT in turn.

As is shown in figure 2(A), we acquired the coding sequences of AFB1-ScFv fusion protein and AFB1-ScFv (nonsp) fusion protein. Also, we added biobrick prefix and suffix to the biobrick through specific primers. How we acquired the coding sequence of SH3 domain-LGT is shown as figure 2(B).

In figure 2(C), the sizes of the aiming stripes are about 900bp, 850bp, 1200bp, which are agree with the sizes of the AFB1-ScFv, AFB1-ScFv（nonsp）, SH3 domain-LGT coding sequences. It can thus be seen that the sizes of the stripes are correct. After sequence analysis, we confirmed that our new biobricks are successfully constructed.

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Implementation of Device

fig.3: (A) From top to bottom，the four figures are the schematic layout of the AFB1-ScFv device, the AFB1-ScFv (nonsp) device, the SH3 domain-LGT device and the final device of the project. (B) The agarose gel electrophoresis of the biobricks group (AFB1-ScFv) of our project. The first lane is DNA marker（the length of the stripes is shown in the figure). The second to the fifth lane are AFB1-ScFv，B0030-AFB1-ScFv，B0030-AFB1-ScFv-double terminator, pbad-ScFv(whole device) in turn. (C) The agarose gel electrophoresis of the biobricks group (AFB1-ScFv (nonsp)) of our project. The first lane is DNA marker（the length of the stripes is shown in the figure). The second to the fifth lane are AFB1-ScFv（nonsp），B0030-AFB1-ScFv（nonsp），B0030-AFB1-ScFv-double terminator（nonsp）,pbad-ScFv（nonsp）(whole device) in turn. (D) The agarose gel electrophoresis of the biobricks group (SH3 domain-LGT) of our project. The first lane is the DNA marker（the length of the stripes is shown in the figure）. The second to the fifth lane are SH3 domain-LGT，B0030-SH3 domain-LGT，B0030-SH3 domain-LGT-double terminator, pbad-SH3 domain-LGT(whole device) in turn. (E) The agarose gel electrophoresis of the final device of our project. The first lane is DNA marker（the length of the stripes is shown in the figure). The second lane is pbad-ScFv-pbad-SH3 domain-LGT.

The brief introduction of the biobricks we have used is shown in table 1.

In figure 3(A), we briefly introduced the composition of our device. For the first three devices on the top, we initially connected the RBS with coding sequence. Then we connected the ligation product with double terminator. Finally, we added a promoter to the beginning of the whole device. The last device in the figure is a ligation product of AFB1-ScFv device and SH3 domain-LGT device.

As is shown in figure 3(B),the sizes of BBa_K1260000 (AFB1-ScFv)，BBa_K1260003 (RBS-ScFv), BBa_K1260004 (RBS-ScFv-double terminator)，BBa_K1260005（Afb1-ScFv whole device）are approximately 950bp，1000bp，1100bp and 1200bp respectively, which coincide with the design of the biobricks.

As is shown in figure 3(C),the sizes of BBa_K1260001(AFB1-ScFv（nonsp）)，BBa_K1260006(RBS-ScFv（nonsp）), BBa_K1260007(RBS-ScFv（nonsp）-double terminator)，BBa_K1260008（AFB1-ScFv（nonsp） (whole device）are approximately 850bp，900bp，1000bp and 1100bp respectively, which coincide with the design of the biobricks.

As is shown in figure 3(D),the sizes of BBa_K1260002(SH3 domain-LGT)，BBa_K1260009(RBS-SH3 domain-LGT),BBa_K1260010(RBS-SH3 domain-LGT-double terminator)，BBa_K1260011（SH3 domain whole device）are approximately 为1200bp，1200bp，1400bp，1600bp respectively, which coincide with the design of the biobricks.

As is shown in figure 3(E), the size of BBa_K1260012 (Afb1-ScFv-SH3 domain-Lgt whole device) is approximately 2800bp, which coincides with the design of the biobrick.

From the above analysis we can confirm that all of our electrophoresis results were correct. After sequences analysis, we confirmed that all of our devices have been connected successfully.

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Function Test

Protein Expression and Inducing Condition

In order to certify that the biobricks we constructed can express the protein we want as expected, we decided to do the western blot experiment. The principle of the experiment is to utilize the 6*his-tag on the protein structure. We used an anti-his antibody with an HRP to bind it and to examine it.

fig.4: The Result of Western Blot Experiment

The result of the first western blot experiment， the first lane is marker (the length of the stripes is as shown in the figure), the second and the third lane is the sample(the chassis is E.coli MG1655) after disposal (Inducted by L-arabinose at 0μM and 8μM separately in turn for 1 hour and then was added 5＊SDS-page sample buffer. After that the sample was heated at 98℃ for 10min separately in turn.) (B) The result of the second western blot experiment， the first lane is marker (the length of the stripes is as shown in the figure), the second to the seventh lane is the sample(the chassis is E.coli MG1655) after disposal(Inducted by L-arabinose at 0μM,0.5μM,1μM,2μM,4μM,8μM separately in turn for 1 hour and then was added 5＊SDS-page sample buffer. After that the sample was heated at 98℃ for 10min separately in turn.)

First of all, we set a simple L-arabinose induction ladder（0μM，8μM）, according to which the sample is processed. After that we started the western blot experiment. The result is shown in figure 4(A). The specific stripe did appeared in the lane, and its size is correct. From that we have confirmed that the target protein exists in our sample.

After the first experiment, we have proved that our samples contain the target protein. In order to explore the best protein express condition, we set a more complicated induction ladder （0μM，0.5μM，1μM，2μM，4μM，8μM）and then we began the second Western blot experiment. In the figure 4(B), the result of the second western blot experiment was shown.

We can see that the specific stripes in the second lane to the fourth lane are observed. The sizes of them are approximately 33KDa, which is coincide with the design of AFB1-ScFv. From that we can prove that our engineered bacteria can express our target fusion protein under the condition of 0μM-2μM concentration of L-arabinose.

Besides, the stripe in the third and the fourth length are broken. The reason why may be the quality of the gel. From the fifth lane to the last lane don’t have any stripe. The reason might be that the growth of the bacteria is inhibited by the L-arabinose. Also, it might be that the AFB1-ScFv is a kind of secreted protein. After inductor increased, the protein was accumulated excessively and it does harm to the cell itself. So the cell produced inclusion body to wrap the protein.

Protein Function Examination and Quantification

After the expression of target protein is confirmed, we began to do ELISA to quantify it. (The schematic diagram is shown in figure 5(A)

The principle of ELISA: Bound the antigen on solid phase. (Maintain its immunocompetence). Then start a reaction between antibodies, enzyme labeled antibodies and the antigens in turn. Then wash away the unwanted substance( the complex substances of antigen and antibody in the solution.). After that, we use a plate reader to detect the luminous intensity. At last we get the quantity of antibodies through calculation.

We used competitive ELISA and direct ELISA， and the analysis of the result is shown below.

fig.5: The schematic diagram and the result of ELISA experiment. The schematic diagram of ELISA: A1 is the standard group. A2 is the competitive group. A3 is the direct experiment group. The AFB1-ScFv is mixed with AFB1 solution before the experiment. When the AFB1 appears in the solution, both AFB1-ScFv and standard AFB1 antibody will combine with the AFB1. Once the AFB1 in the solution is bound by any antibody, the antibody won’t be able to combine with the coated AFB1. The result of that is ：As the AFB1 in the environment increased，the lightness will be decreasing. In the direct experiment group, the situation is different. The AFB1-ScFv will directly combine with the coated AFB1. The more AFB1-ScFv there is( within a certain range)， the brighter the sample will be. (B) The result of competitive ELISA experiment. (C) The result of direct ELISA experiment.

We made the standard curve, and examine whether the AFB1-ScFv is working or not though changing the concentration of AFB1 in the solution. The result is shown in the figure 5(B).

As the result of the experiment is shown, the coated AFB1 antigens bounded by the AFB1-ScFv are less than those which are bounded by the standard AFB1 antibody. From that we can get the conclusion: The AFB1-ScFv expressed by our engineered bacteria can combine the AFB1. The lines in the figure are not paralleled. We considered it as the result of the lack of the target protein. After the examination by SDS-page electrophoresis, we confirmed that the reason is true.

 Besides, when the concentration of AFB1 reaches 1ng/ml, the quantity of AFB1-ScFv that bounded with AFB1 in the sample is nearly zero. The reason may be that under high concentration condition of AFB1 in liquid phase, the combining weight of AFB1 and AFB1-ScFv is nearly zero so the machine can’t detect the luminescence.

Also, we found out that the sample with broth will increase the amount of Aflatoxin B1 (which is impossible). We consider it as systematic error and we will repeat the experiment. But we didn’t get ideal result because the time is limited.

In the direct group experiment, the situation is different. The AFB1-ScFv will combine with the antigen directly. So the more AFB1-ScFv there are, the brighter the sample will be. The result is shown in the figure 5(C). The result shows that while the induction concentration is rising, the brightness is increasing too So we can prove that the AFB1-ScFv has the tendency that the quantity of it will rise while the induction concentration rising.

Positioning of the AFB1-ScFv

The function of bringing the antibody back to the membrane has been designed， so we can make the AFB1 tight on the membrane. We can use the method to examine the function shown below:

Though co-Immunoprecipitation. After the disposal, the SH3 domain and AFB1-ScFv will be connected (The essential precondition is that they have interaction.) Through western blot examination we can get a specific stripe of 77KDa. And that can prove that we have achieve our goal.

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Discussion

After the two examination experiment, we have proved the our engineered bacteria can finish the task we give to them.

We have examined through the western blot experiment that our engineered bacteria lysate has target protein in it, and the expression quantity increased with the induction concentration(though it is not obvious). But the amount of soluble protein didn’t increase with higher induction concentration (figure 4)

The reason might be that the growth of the bacteria is inhibited by the L-arabinose. Also, it might be that the AFB1-ScFv is a kind of secreted protein. After concentration of induction increased, the protein accumulated excessively and it does harm to the cell itself. So the cell produced inclusion body to wrap the protein.

The data of ELISA shows that our fusion protein bound with AFB1 successfully. (Besides, under high AFB1 concentration condition, the quantity of AFB1-ScFv that bounded with AFB1 in the sample is nearly zero. The reason may be that in high concentration of AFB1 in liquid phase, the combining weight is nearly zero so the machine can’t detect the luminescence.)

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Future Work

After analyzing the results mentioned above, we have rudimentarily implemented our project, but the results were not good enough. So we will begin to improve the project.

Improvements on the circult

1. We will continue our positioning experiment to find out whether the membrane protein can be dragged to the membrane.

2. We will carry out fluctuation analysis and modeling. In addition, we will change some parts of the circuit to figure out the best condition to produce artificial protein and to increase its production.

3. We will change the temperature and moisture condition and change the Aflatoxin content in the environment to certify the bacteria’s function to reduce Aflatoxin.

4. We will change the trigger of this experiment to an automatic fashion to satisfy practical needs in real life.

5. We will add more coding sequence for additional functions to the circuit, such as bacteria killing and further process of the toxin.

6. We will add a suicide device to the circuit so that the bacteria will die if their quantity reaches a lethal level or if gene mutation occurred.

7. We will refine and decorate the protein and increase its efficiency to bind Aflatoxin B1.

Applications

Because many farm products such as groundnut oil and vegetable oil are made directly from botanic objects and crops, the Aflatoxin quantity of them may easily reach a fatal level. Therefore our project discovers and provides a new approach to solving such problems, which is a global pollution concern. Fermentation and industrial usage should also be considered. For instance, cultivating the bacteria in a semi-permeable broth to filter milk or oil, or cultivating them on a solid medium with crops shaking above is great feasible practice. Single-stranded antibody can be replaced by other kind of antibody. With the similar ligand-domain system, any substance with correspondent antibody can be immobilized on the bacteria membrane. We will conduct social surveys on our project, as well as consulting relevant scholars and experts. We will improve our project and solve its remaining problems with the suggestions and advice from the public and the professionals.

Part	ID	Introduction
Pbad weak promoter	BBa_K206001	This is a pbad weak promoter. We use it to realize the artificial control function.
RBS	BBa_B0030	This is a strong RBS., We use it, in order to obtain a high expression of protein.
Terminator	BBa_B0015	This is a double terminator with strong termination.
Backbone	psb1C3	The requirement of the iGEM community.
CDS	BBa_K1260000, BBa_K1260001, BBa_K1260002	The plasmids constructed in the project.