Team:TU-Delft/PeptideCharacterization

From 2013.igem.org

(Difference between revisions)

Revision as of 12:37, 2 October 2013

Peptide Characterization

An important part of the project is inhibition of growth or killing of bacteria with the use of antimicrobial peptides (AMPs). In order to get an idea of the toxicity of our peptides we conducted several minimal inhibiting concentration (MIC) experiments. These MIC measurements where done on E. coli, B. subtilis andS. delphini, with the first as representative of our Gram-negative expression host, the second for the Gram-positive targets and the last for our specific target. The AMPs Signiferin, maximin H5 and magainin were chosen from literature [1,2,3] as being active against staphylococcus species but not against E. coli in order to able to use the latter as the expression host. MICs were also determined for the novel peptides we designed as described ( here).

Procedure

Grow the appropriate strains overnight and make a 1/50 to 1/100 dilution the next morning and wait for the OD at 600nm to reach 0,4-0,6.
Make a 100µM stock of the AMP in LB medium, this solution will serve as a 100 X stock, as the expected MIC range of our AMPs lays between 5 and 40 µM.
Then make the combinations as shown in table 1 in a 96 well plate, for the plate reader to take readings using an plate reader capable of shaking and heating to 37˚C. Taking measurements every 10 minutes.

	No cells + 1mM	40µM	30µM	25µM	20µM	15µM	10µM	7.5µM	5.0µM	2.0µM	1.0µM	Cell + No AMP
LB(µL)	90	55	65	70	91	75	80	85	87.5	90	93	94
Cells(µL)	-	5	5	5	5	5	5	5	5	5	5	5
100μΜ(µL)	10	40	30	25	20	15	10	7.5	5.0	2.0	1.0	-

Results

MIC determination

The MIC of signiferin on S. delphini, B. subtilis and E. coli were done according to the protocol described above. S. delphini is the most sensitive for signiferin, as the MIC was determined to be 1µM (Figure 1A), no growth is seen at this concentration. The MIC for B. subtilis was determined to be 10µM (Figure 1B).

Figure 1: MICs of Maximim-H5

The expected selectivity of the chosen peptides for Staphylococcus was confirmed by these experiments, as for all the peptides for which we could determine a MIC, that MIC was the lowest on S. delphini. Maximin H5 and magainin did not give a measurable reduction in growth below 40µM (Figure 2), making us decide not to proceed testing, as modelingshowed it was not possible to reach these concentrations through expression in E.coli.

Figure 2: MICs of Maximim-H5 and Magainin

The fact that for none of the peptides a MIC could be determined for E. coli (>150µM) further confirms the expected selectivity towards Gram-positives (Figure 3).

Figure 3: MICs of Maximim-H5, Signiferin and Magainin in E.coli

Of the novel peptides two were determined to be active against S. delphini; sebastini and joepini, with MICs of respectively 30 and 40µM (Figure 4, Figure 5). .The MIC of sebastini on B. subtilis however was determined to be <10µM (Figure 5B) lower than that for S. delphini. As for the peptides described above, no MIC could be determined for E.coli (Figure 6).

Figure 4: MICs of JoepininMaith

Figure 5: MICs of SebastiniDim

@@ Line 280: / Line 280: @@
 <br>
 </p>
-<h4 align="left">COS-1 toxicity determination </a>
+<h4 align="left">COS-1 toxicity determination </h4>
 <p align="justify">
 In order to determine the toxicity of the AMPs on mammalian cells we tested the most promising peptides on COS-1 cells <a href="https://2013.igem.org/Team:TU-Delft/PeptideCharacterization#references">[4]</a>. As signifirin, sebastini and joepini gave the best results in the experiments described above they were chosen to be tested with concentrations up to 150µM on COS-1 cells. COS-1 cells were chosen as they, because of their simian nature, strongly resemble human cells with respect to membrane properties and crucial cell functions and mechanisms <a href="https://2013.igem.org/Team:TU-Delft/PeptideCharacterization#references">[5]</a>. Normally healthy COS-1 cells attach to the bottom of the well, showing a clear fibroblastic morphology <a href="https://2013.igem.org/Team:TU-Delft/PeptideCharacterization#references">[6]</a>. When they start to die they will swell and eventually detach from the bottom forming spherical cells which upon lysis leave behind cell debris.