From June 14th to 23rd
We performed the inhibitory activity of the bacteriocin Nisin and Lysozyme. According to:
Inhibitory assays for S. aureus, E. coli, P. aeruginosa, E. faecalis and L. lactis were performed. We followed the protocol according to Barboza-Corona et al. (2007). For Nisin, eight dilutions 1:1 were made with 50 mM citric acid with 50% ethanol, which were placed on the wells (100 μL) and the highest concentration was 250 u/ml. As negative control was used 50 mM citric acid only. For Lysozyme, a 1 mg/ml stock was used and this was the highest concentration. Also eight dilutions were made with distilled water and no negative control was performed.
The growing media for each bacteria was Tryptic Soy Broth (TSB, 0.7 g) plus 6 g of agar for 100 mL.
We checked the construction for LasR gene to see which enzymes were required for the cutting and we designed the oligos for this gene.
We did the dilutions for Nisin and Lysozyme and we performed all the protocol for the in vitro inhibitory activity.
We finished the last part of the protocol for the inhibitory activity and we designed the oligos for the RhIR promoter. The enzymes required were EcoRI and BamHI.
We did the inhibitory activity again because of contamination. We did new dilutions of Nisin without ethanol.
We performed the protocol for biofilm formation of P. aeruginosa, according to Toodle (2011).
We performed the Lysozyme extraction from human blood using the protocol for RNA Extraction with TRIzol® according to Invitrogen.
We did new dilutions with Nisin now diluted in distilled water because it gave us better results (data not shown). We also runned the gel for checking the quality of the RNA extracted. The gel was 1.2% Formaldehyde Agarose, ran at 1 hour at 90V, we do not observed anything because the time was too much.
We did a new electrophoresis gel was 1.2% Formaldehyde Agarose, ran 20 minutes at 90V, we observed only a one little band because the buffer was very strong.
We performed the Lysozyme extraction from human blood using the protocol for RNA Extraction with TRIzol® according to Invitrogen. We did a electrophoresis gel was 1.2% Formaldehyde Agarose, ran 5 minutes at 90V, 15 minutes at 60V, 30 minutes at 49V. The samples were degraded.
We performed the amplification by a PCR reaction of the signal peptide ChiA from B. thuringensis, the promotor RhIR from P. aeruginosa and Alginate lyase from B. thuringensis.
We did another PCR reaction for the signal peptide (SP) for us to have a duplicate and it was the only successful reaction. We performed the Lysozyme extraction from human blood using the protocol for RNA Extraction with TRIzol® according to Invitrogen.
We did a denaturant electrophoresis gel to RNA of human lysozyme samples. We do not observed anything because the gel had very much Bromure Ethidium.
We did the transformation of the pCOLD vector with (TOP10 Cells), Following next procedure:
We did a electrophoresis gel was 1% Formaldehyde Agarose and prepared the electrophoresis camera with NaOH 0.1N for 1 hour, the water and buffer are prepared with DEPC. We load two samples of RNA of human lysozyme. We observed three bands, one for DNA and other two for RNA 16S & 32S.
Add into the electroporation cell 100 μL of E. coli TOP10 + 1μL of the ligation product.
Perform the electroporation at 2.5 kV.
Add immediately 1 mL of liquid LB without antibiotic into the cell after electroporation.
Resuspend by gently mix
Incubate for 1 hour at 37°C
Cultivate 100 μL of the bacteria in a Petri dish.
Centrifuge 1 min at 9,000 rpm.
Pour the supernatant and leave approximately 100 μL. Resuspend by pipetting.
Cultivate another Petri dish with this concentrated cell culture.
We performed the DNA extraction of the pCOLD according to Jena Bioscience, and we also performed the purification of SP by the PCR Purification Kit by the same company. We keep the ligation of pCOLD + SP overnight.
We made treatment for the ARN with DNAse and EDTA, after made a RT PCR with iScript protocol by Invitrogen. We also runned the gel for checking the quality of the DNA amplified. We do not observed anything.
We keep the ligation reaction on the thermoshaker until it reaches an approximate volume of 5 μL. We performed the dialysis procedure for this ligation DNA. We did the transformation of E. coli in order to introduce pCOLD +SP. We repeat the RT PCR and used a temperature gradient at 50° C to 60°C.
We performed the amplification of pCOLD + SP by PCR reactions and then checked it in an electrophoresis gel.
Since we did not obtain a good result of the amplification we repeated the entire protocol of ligation starting with the digestion of SP with BamHI and KpnI. We did the ligation with the same quantities of components. We used a Isolation of mononuclear cells by density gradient separation with Ficoll-hypaque for isolate human leukocytes, but we do not had a good results.
Finally we try to continue work with the older samples and repeat the RT PCR with iScript, but do not obtained bands in electrophoresis gel, and decided discard the Lysozyme and try with other compound.
The grown colonies were used for growing them into liquid LB medium in Falcon tubes. In total 24 colonies were grown. The tubes were kept overnight. In addition, we prepared the solutions required for the protocol, Isolation of DNAp for use in sequencing. We inoculated 5 strains that possibly possess the alginate lyase gene, B. thuringiensis 4Q7, B. thuringiensis HD73, B. thuringiensis HD1, B. thuringiensis Cry-B, B. Cereus.
We transferred 1-1.5 μL of the medium in the Falcon tubes into Eppendorf tubes for making the extraction of DNAp. we extracted DNA for the cultures possibly possess the alginate lyase gene using a boiled extraction protocol. After we make a PCR for isolate alginate lyase gene, ran the samples at electrophoresis gel and we observed positive bands for B. thuringiensis 4Q7 and B.cereus.
We retried the digestion of sp+pCOLD with BamHI y Kpn1.
We purified alginate lyase and pCOLD with the Kit PCR Purification of Jenna Bioscience, considering five volumes of binding buffer.
We transformed TOP10 cells with alginate lyase DNA. following the standard protocol.
We proceeded the digestion of pCOLD+sp, and ligation of pCOLD+Alginate lyase.
We did the PCR of DNa from pCOLD+sp, this day we confirmed 4 colonies, with the protocol TAQ THUR. Then the product was purified.
We confirm a one positive colony with the digestion with PstI y BamHI. In addition we quantified the product of sp. The same day we put the triple ligation of sp + alginate lyase and pCOLD.
We proceeded to transformed the ligation, perhaps until 29 th, we tried the ligation with non positive result.In addition we produced TOP10 cells with the respectivily protocol.
This month we paused the development of molecular assays, only a few days preparing material to complete the experiments.
We tried the triple ligation, and additional work for modelling and project.
The first two weeks of the month, we worked on the submission of our parts.