Part:BBa_K875009:Experience

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Applications of BBa_K875009

Improvements: improved part: K1892000 Edited by Yingqi Wang, 2016 SMS_Shenzhen team:

We improved the part BBa_K875009, human antimicrobial peptide LL-37, by fusing it with a motor protein yebf (The improved part is BBa_K1892004：https://parts.igem.org/Part:BBa_K1892000). Therefore, LL-37 can be carried out through cell membranes by yebf and is much more convenience for use. We have also proved with our experiments that BBa_K1892004 is effective against Gram-positive and Gram-negative bacteria, thus to characterize BBa_K875009 further.

Antimicrobial Effect of LL-37: To test the wide anti-bacterial function of LL-37 expressed by E.coli, we selected three commonly found representative bacteria including both Gram-positive and Gram-negative Bacteria: Staphylococcus aureus, Bacillus subtilis, and the not genetically modified Escherichia coli, using the LB culture for cultivating the bacteria. First, we spread the liquid bacteria on the LB solid in petri dish. After the film of liquid bacteria is completely dry on the LB solid culture, we separate the petri dish into four parts as the diagram below shows.

Figure 1: diagram for experiment design

In this experiment, water is the negative control and antibiotic carbenicillin as the positive control. On each section, we add a drop of 10μL of the corresponding liquid. After all the liquid is dry, the petri dishes are placed in 37℃ incubators for cultivation. We set up comparisons as well, one section is added with induced yebf-LL-37 as the experiment group; another one is added with yebf-LL-37 that had not been induced as the control group.

The results:

Figure 1. Antimicrobial effect of yebf-LL-37 against Bacillus subtilis

Figure 2. Antimicrobial effect of yebf-LL-37 against Bacillus subtilis

Figure 3. Antimicrobial effect of yebf-LL-37 against E.coli

The results matched our expectation and proved our antimicrobial concept. For all three types of bacteria, yebf-LL-37 that had not been induced did not produce an antibacterial effect while ampicillin is effective. For the part added induced yebf-LL-37, we can see a clear circle in the middle of each section with induced E.coli, which mean that LL-37 effectively inhibited bacterial growth in B.subtilis and S.aureus. The effect of it in E.coli is not so obvious, which may due to that the chassis is E.coli. From this experiments, we proved that the antimicrobial peptide LL-37 is effective against both Gram positive and Gram-negative bacteria.

User Reviews

UNIQa1282de3075409f1-partinfo-00000000-QINU Team Ciencias-UNAM 2013 ( http://2013.igem.org/Team:Ciencias-UNAM ) used this part as a part of its project. Given Trieste Team couldn't characterise this part properly, we analysed the problems with theirs characterisation to express this protein properly.

They used Lac promoter to express this peptide, hoping to inhibit cell growth. They did not have positive results, as the control experiment growth the same way as the one with LL-37 expression with IPTG. We approached this problem with two solutions. The first problem we saw in Trieste's characterisation was that Lac promoter has a very high basal level of transcription. Given pSB1C3 has a high copy number, it is transcribing a high amount of LL-37, still with no IPTG in the media. This could mean that there is selection pressure on E. coli to loose the plasmid, which could be one of the reasons the did not see changes between the control cells and the cells under IPTG induction. We addressed this problem by changing the promoter, constructing device BBa_K1230007. This device is under the pBad promoter, which works with arabinose. This promoter has a basal level of almost 0, so we are sure that it is not transcribing any LL-37 that could be making the cells to loose the plasmid. The second problem we addressed was the change in the codon usage they used. By changing the codons to produce more peptide in E. coli, the peptide may not have sufficient time to fold properly, altering it's function. We solved this problem by using the normal codon usage of this peptide, allowing it to fold properly into it's final conformation.

For the characterization of the LL-37 antimicrobial peptide from Trieste, with another promoter, we inoculated 12.5 ml of LB of the strain without the plasmid, and the strain with the plasmid (we made pressure selection with chloramphenicol to make sure our plasmid was still there). We did the inoculation at 0.05 of OD at 600A and incubating at 37°C until it got to 4.0 OD at 600A. Then we separated each of the two tubes in 5 15ml falcon tubes, 2 ml of the inoculums. We induced with arabinose as we mention in the following table. From each tube we did 8 decimal dilutions in 1.5 Eppendorf tubes with LB. These are the results.

As we can see here, there is no basal level transcription on this experiment. So the problem in theirs characterisation may be due to the codon usage they used.

For more information on the characterisation, visit our notebook on our wiki http://2013.igem.org/Team:Ciencias-UNAM/Project/WetLab/Protocols UNIQa1282de3075409f1-partinfo-00000001-QINU