Part:BBa_K1647020:Experience

Function Testing Experiments

1. Turbidimetry

To test the activity of AAP2 and Bac8c under different pH conditions, we conducted these 2 experiments below with a 722s spectrometer.We cultured the two kinds of E.coil (BL21), one containing the plasmid Pet28a-AAP2 and the other the plasmid Pet28a-Bac8c, in the liquid Luria-Bertani media.

A. Effect of AAP2 on E.coli under different pH

After being cultured to OD=600, E.coli containing Pet28a-AAP2 are induced with 1.0mM IPTG and the pH of the bacterial solution is adjusted to either pH=5 or pH=7 by adding hydrochloric acid. Then the OD of the solution is analyzed every 30 minutes, and the result are as follows:

From the picture we can see that with the induction of IPTG, E.coli’s growth is strongly inhibited after 5.5 hours, indicating that AAP2 is an effective antimicrobial peptide. However, the inhibition effect of AAP2 are similar between groups pH7 and pH5, indicating that AAP2 may not be an acid activated antimicrobial peptide. Nevertheless, considering that the pH of the external environment is much easier to change than that of the inner cell environment (because of the homeostasis of E.coli), and since AAP2, after induction, is expressed and takes effects inside E.coli, we can’t say that the pH change of the external environment will indeed affect the structure of AAP2 inside E.coli, and thus we can’t deny it that AAP2 may have stronger inhibiting effect at lower pH. Further experiment should be conducted to prove our guess, where the expressed AAP2 will be purified and artificially added to an E.coli culture, so that the AAP2 can take effect from the outside of the cell. However, according to the result we get, it’s one thing for sure that AAP2 has antimicrobial effect under pH7.

B. Effect of Bac8c on E.coli under pH=7.0

After being cultured to OD=600, E.coli containing Pet28a-Bac8c are induced with 1.0mM IPTG and the OD of the solution is analyzed at 0, 1, 3, 7 hours after induction. The result are as follows:

From the picture we can see that with the induction of IPTG, E.coli’s growth is strongly inhibited after 7 hours, indicating that Bac8c is an effective antimicrobial peptide.

2. SDS-PAGE Electrophoresis

To detect the antimicrobial peptides expressed by E.coli (BL21), we collected 70mL sample from each group of the Turbidimetry experiment above in the 240min. Then we lysed the samples with a ultrasonic Processor, getting 3 kinds of bacterial lysis solutions: • Pet28a-AAP2, pH=7 • Pet28a-Bac8c, pH=7 • Pet28a-CAP-Bac8c, pH=7 With the 12% separating gel and the 3% stacking gel;we had our samples with loading buffer run for 20min in stacking gel under 120V and 60mim in separating gel under 100V.

As in the picture, the band of the three antimicrobial peptide AAP2, Bac8c and CAP-Bac8c are really vague and unclear,even we cannot give a label. However the samples were also tested for the Kirby–Bauer antibiotic testing, which confirmed the existence and the function of our peptides. So here are several possible reasons: The relative molecular mass of those peptides are no more than 2kD, which is very difficult to dis-tinguish on the gel. Besides the band intensity of other proteins are very light, indicating that the concentration of our sample may be very low, making it even harder to identify the band of antimi-crobial peptide.

3.Kirby–Bauer antibiotic testing

To confirm the existence and the function of our antimicrobial peptides, we conducted the experiment with the bacterial lysis solutions collected earlier :

• Using an aseptic technique, place a sterile glass rod onto the culture of E.coli and then gently streak the Mueller-Hinton agar plate to form a bacterial lawn.To obtain uniform growth, streak the plate with the glass rod to and fro, meanwhile rotate the plate 90° and streak the plate again and again. • Allow the plate to dry for approximately 5 minutes. • Using an Antibiotic Disc Dispenser to dispense discs containing our antimicrobial peptide and also sterile water onto different plates. • Using a flame-sterilized forceps, gently press each disc to the agar to ensure that the disc is at-tached to the agar. • Plates should be incubated overnight at an incubation temperature of 37 °C for 16 hours. • Observing and comparing the zones of inhibition in different plates.

The positive control group was the 75% ethanol while the negative control group was the sterile water. After culturing the bacteria for 16 hours, we got the results below:

Figure： A.pET-AAP2 (pH=5) B.pET-Bac8c (pH=5) C.75% ethanol (left)/ sterile water (right) D.pET- AAP2 (pH=7) E.pET-CAP-Bac8c (pH=7)

This picture shows different kind of antimicrobial peptide we designed used in Kirby–Bauer antibi-otic testing. Through this, we could find that comparing with negative control (sterile water) in plate C, even not that clearly, the diameters of zones of inhibition are larger. Because the antibacterial effect of ethanol is not prominent, the diameter in plate C are small. Comparing with plate A and D, because AAP2 should have stronger effect in low pH, and the re-sults show that the diameter in A are larger than D, means in low pH environment, the AAP2 are more effective. Comparing with plate B and E, we test whether CAP may have influence on bacteriostasis of Bac8c, as we thought before, CAP have a special function on location Streptococcus. mutans, the precondition is CAP cannot interfere Bac8c. And this picture shows the future of orientation of an-timicrobial peptide, that is, the existence of CAP do not influence the diameter of the zone of inhibi-tion. Our experiment have some flaws, however, we can make a simple conclusion that, through the experiment, we find these antimicrobial peptide do have the bacteriostasis, even in different condi-tion, they can work.

Conclusions

We tested the functions of our antimicrobial peptides in E. coli through Turbidimetry, SDS-PAGE Electrophoresis, Kirby–Bauer antibiotic testing these 3 groups of experiments. And from them we can confidently conclude that: • All the peptides can be expressed correctly and with expected functions in E.coli (BL21); • The Bac8c is more active under pH=7 while AAP2 is more active under pH=5; • The peptides with CAP guiding part are stronger the the ones without it.

Future Expectations

Unfortunately we didn’t get to test our antimicrobial peptides on the S.mutans so we hope that we could conduct more tests and collect more data in the future with a system based on S. mutans, our primary target. What’s more we hope that we could transfer the plasmids into the S.mutans so the antimicrobial peptides couid be delivered directly into the biofilm on the surface of the teeth that may suffer dental caries. This means we need to find other suitable plasmids and some more environmental sensitive promotors.

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