Team:UI-Indonesia/Parts/Characterization

Peptide 1018 Antimicrobial Assay

Background
Peptide 1018 is small cationic peptide under T5 promoter (inducible promoter). Peptide 1018 in particular concentration can be toxic for bacterial vector. Isopropyl thiogalactoside (IPTG) is analogue of lactose that is able to induce activation of T5 promoter to start transcription.

Aim
To examine in which concentration of IPTG that can be toxic for E.coli as bacterial vector

Result



Discussion
From the experiment above we expected that IPTG induction will activate peptide 1018 trancription which is able to inhibit the growth of E.coli and can reduce the number of cells if the cells grow together with IPTG in the start point. In this experiment we the activation was conducted when OD 0.1 and dilution 1:20 without incubation before. The data above show no inhibitory effect of IPTG as increase the concentration. But when we measured the OD600, all treatment show lower in OD600 of IPTG 0 mM. We assumed that the peptide 1018 still working, even though we need more data and incubation period before treated with IPTG.

Starch Hydrolyzed Assay Using Iodine Test

Background and Aim
We designed an E. coli having inserted MalS and HlyA gene in pSB1C3 plasmid in E.coli Top10 BBa_K13440004 (MalS HlyA in pSB1C3), and E. coli having plasmid inserted MalS and HlyA gene in pSB1C3 under control strong prommoter constitutive 100 BBa_K13440005 (strong constitutive promoter J23100-MalS-HlyA in pSB1C3) and E. coli Top10 wild type as negative control were growth on LB starch agar.
Mals is gene that has responsible to encode the enzyme expression of alpha amylase for E. coli strain K12, the length is 1967 bp. The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). Fusion of the HlyA signal peptide to the target protein (MalS) cause the excretion of protein to extracellular medium in a single step.

Methods
Upon successful cloning of the three genes into our E.coli, we continued to confirm that all three genes are required have hydrolizing amylum activity on LB starch agar. We using iodine test to staining the amylum, and see the clearzone for some time variables.

Result

Discussion
We conducted starch hydrolyzed assay using three supernatant sample of E.coli on LB agar starch the flooding iodine on it.. We had two E. coli mutant, they are MH bacteria ( MalS and HlyA gene in pSB1C3 plasmid) and SMH bacteria (strong promoter constitutive-MalS-HlyA in pSB1C3), then we use E. coli Top10 wild type as negative control were growth on LB starch agar.
From the Assay result on 60 hours we can see if there were significantly different, SMH have the widest clear zone, next MH and E.coli wild type have the narrowest clear zone. SMH bacteria have the widest clear zone because the bacteria have an ability to secrete the enzyme into supernatant. But in MH bacteria they are enable to secrete enzyme and up take amylum as carbon source. On E.coli supernatant, the floating cell on supernatant aggregate become e new colony and growing on LB starch agar without have any clear zone or starch hydrolyzed activity

Alpha-Amylase and Nuclease Characterization in Biofilm Removal

Background
Biofilm as matrix extracellular polymeric substances (EPS) causes an increase in antibiotic resistance and pathogenecity of pathogenic bacteria.

Aim
We present our Genius E. coli agent that can degrade EPS biofilm by secreting enzymes, such as alpha amylase and nuclease.

Result

- Produced an E. coli strain which can degrade biofilm matrix
- Demonstrated that E.coli can secrete amylase to hydrolyze amylum using iodine test on the supernatant
- Identified experimentally a potential of enzyme extracellular secretion into supernatant using HlyA tag to produce active enzyme.




We read the absorbance of the biofilm assay by using ELISA Reader model 680 microplate reader in wavelength 595 nm. Then we make the data averages and performed into a diagram bellow:

After calculate the average value of each treatment, we want to compare the data whether the data significantly different by using SPSS 16.00 analysis to find p value. Then we do data distribution analysis by using Saphiro-Wilk test.
From the Saphiro-Wilk analysis we find the p is less than 0.05, it is indicated that our data have normal distribution. After that, in order to support the hypothesis we continue to do Independent T-test analysis.

Note:
*: indicate the data were significant result (data show significantly different if p value < 0.05); red color indicate the most effective enzyme degrading biofilm matrix
MH : MalS-HlyA; SMH : S100-MalS-HlyA; NH: Nuc-HlyA; SRNH: S100-RBS-Nuc-HlyA
Mix: MalS-HlyA and S100-MalS-HlyA placed together in same LB broth

Discussion
Less absorbance value indicate more effective the action of enzyme degrade biofilm matrix. From the data above we focus on data strong constitutive promoter-RBS-MalS-HlyA and MalS-HlyA device. From data above show that α-amylase is more effective degrading Pseudomonas aeruginosa. α-amylase compared to no treatment shows significant result in degrading biofilm matrix (p<0.05). This is due to polysaccharides compound is greater in Pseudomonas aeruginosa. This data is consistent when the addition of α-amylase and nuclease together in LB broth, it is also show significant result in degrading biofilm matrix. But α-amylase less effective in degrading Vibrio cholera, Bacillus substilis, Staphylococcus aureus, Klebsiella pneumonia, and E.coli. We expect that the component of polysaccharides in those bacteria is lesser than in Pseudomonas aeruginosa. Besides that the mutant E.coli need more time to degrade the biofilm because we only incubate 5 hours 37oC. Another factor that make the data error is there were crystal violet left when crystal violet washing.
We compare second device, Strong constitutive promoter-RBS-Nuc-HlyA (SRNH) with no treatment of E.coli mutant, the result shows that, SRNH treatment is more effective in degrading Klebsiella pneumonia biofilm (p<0.05). We expect that extracellular DNA content in Klebisella biofilm is greater than others component. Comparation of mix and no treatment data show almost significant result in biofilm removal. (p=0.058). But we considered that data is significant.
We also can make a conclusion that no promoter and ribosomal RBS can not be recognized by RNA polymerases so the enzyme can not be expressed.