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Team:UI-Indonesia/Parts/Characterization
From 2014.igem.org
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
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 (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 idine 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 are significant differenciate, SMH have the widest clearzone, next MH and E.coli wildtype have the lowest clearzone. SMH bacteria have the widest clearzone because the bacteria have an ability to secrete the enzyme into supernatant. But in MH bacteria they are anable to secrete enzyme and up take amilum 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 clearzone or starch hydrolized activity
