Difference between revisions of "Part:BBa K1260000"

Latest revision as of 17:57, 27 June 2014

AFB1 ScFv with a his-tag, a SH3 ligand and a signal peptide

AFB1 ScFv with a his-tag, a SH3 ligand and a signal peptide. The signal peptide is at the beginning of the sequence. With the part, you can let your E.coli produce antibody to target the aflatoxin B1.

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 orange part is VH.

Result

We use western blot and ELISA to examine the function of the biobrick

Western Blot

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 2(A). The specific stripe did appeared in the lane, and its size is correct. From that we have confirmed that the target protein.

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.

The sample inducted by lower concentration has the same amount of protein and it might be that the AFB1-ScFv is a kind of secreted protein is expressed out of the cell. 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 didn’t appear any stripe. The reason might be that the growth of the bacteria is inhibited by the L-arabinose. Further more, the reason might that 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.

ELISA

fig.3: The schematic diagram and the result of ELISA experiment. A.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 3(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.   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 3(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.

Sequence and Features