Characterization of K1362011
The characterization of R0011 + K1362011 was performed to obtain quantitative results of the action method of the biobrick BBa_K1362011. In order to make the characterization, induction, dilution and antibiogram essays were carried out to determine the lambda lysozyme ability to lyse cells.
Result of gels and descriptions
Figure 1. Electrophoresis of BBa_R0011+BBa_K1362011 PCR and a plasmid digestion of, BBa_R0011 and R0011+K1362011 in a 0.8% agarose gel run in a 1 X TAE buffer at 100 V for 50 minutes and stained with GelRed 0.3X. 1) Quick-Load Purple 2-log DNA ladder diluted 1:3 in MB grade water (NEB) (3 μL + 4μL LB) 2) Digestion of R0011+K1362011 with MlyI (5 μL + 4μL LB) 3)Digestion BBa_R0011 with MlyI(5 μL + 4μL LB) 4) BBa_R0011+ BBa_K1362011 PCR (08/07/2016) (5 μL + 4μL LB) 5) BBa_R0011+ BBa_K1362011 PCR (07/07/2016) (5 μL + 4μL LB)
Figure 2. Electrophoresis in polyacrylamide gel of BBa_R0011+BBa_K1362011 Induction and solubility in a 12% acrylamide, Lambda lysozyme has an expected weight of 17.83 kDa. 1) Empty well 2)Hour 0 (No induction) - 30μL 3) Hour 1 - 30μL, 4) Hour 2 - 30μL, 5) Hour 3 - 30μL, 6) Hour 4 - 30μL, 7) Precision Plus Protein All Blue Prestained Protein Standard (Bio-Rad) - 5μL, 8) Empty well, 9) Soluble phase -20μL, 10)Insoluble phase - 20μL, 11) Precision Plus Protein All Blue Prestained Protein Standard (Bio-Rad) - 5μL.
Figure 3. Electrophoresis in polyacrylamide gel of BBa_R0011+BBa_K1362011 Induction and solubility in a 15% acrylamide, Lambda lysozyme has an expected weight of 17.83 kDa. 1) Precision Plus Protein All Blue Prestained Protein Standard (Bio-Rad) - 5μL. 2) Control BL21 20μL 3)Hour 0 (No induction) 20μL, 4) Hour 1 - 20μL, 5) Hour 2 - 20μL 6) Hour 3 - 20μL, 7) Precision Plus Protein All Blue Prestained Protein Standard (Bio-Rad) - 5μL, 8)Hour 4 - 20μL, 9) Soluble phase - 20μL, 10)Insoluble phase - 20μL.
Table 1. OD results of characterization essay.
Table 2 OD results of characterization essay.
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Figure 5. Dilutions of protein extract with culture of E.coli. a) Control, only LB. b) 200 ul dilution. c) 400 ul dilution. d) 600 ul dilution.
The transformation of R0011 + K1362011 was performed in BL21 competent cells, to assure the plasmid had the biobrick a Colony PCR was made, figure 1 shows the plasmid being amplified by VRF primers, wells 6 and 7 show the correct band amplified at approximately 800 bp. The first induction and solubility analysis (figure 2) could not conclude the presence of the protein because the gel was not the correct percentage for the kilodaltons of the protein (approximately 17 kDa). To obtain conclusive results induction and solubility analysis were repeated and runt in a 15% acrylamide SDS-PAGE (figure 3 and 4). The Characterization essay was performed according our Characterization Protocol, the results are listed in Table 1, but since the OD results were too high the protocol was repeated in order to obtain trustworthy results listed in Table 2. This dilutions were plated in LB plates as we can see in figure 4, 5, 6 and 7, it can be seen that the number of colonies diminishes a lot as we increased the quantity of our protein extract. Antibiograms with the protein extract were made (figure 6), in this figure is show the growth inhibition of J04450 transformed cells near the protein extract, we can see as well a little increase in the inhibition halo as the protein extract increases.
In conclusion after the dilutions and antibiogram analysis of the plates of the R0011 + K1362011 characterization, the values of MIC and MBC could be calculated, the MIC values were concluded as the concentration in which there is no visual growth and the MBC is the lwhere there was almost no growth at all, this values were delivered as MIC of 250uL and MBC of 600uL of proteic extract.
Documentation of Parts
WELK and NNQW are the most important parts of the project, for this we did several protocols just to document this parts. WELK is the holine that helps us destabilize the peptidoglycan layer and NNQW is the composite part of WELK and E3D0 (the holine and the endolysin).
Fig.1 Colony PCRs of the project in general in a 0.8% agarose gel run at 70 min 100V. 1)Empty
well, 2) NNQW Colony 5μL + 4μL LB, 3) E3D0 Colony 5μL + 4μL, 4) WELK
Colony 5μL+ 4μL LB, 5) 2-log DNA LADDER NEB 2μL + 4μL LB,6) TDSH Colony 5μL + 4μL, 7) DO1P Colony 5μL + 4μL, 8) 9A3D Colony 5μL + 4μL, 9)2-log DNA LADDER NEB 2μL + 4μL LB, 10) ELLK Colony 2μL + 4μL LB.
Fig 2. Colony PCRs of WELK 0.8% agarose gel run at 50 min 100V. 1)2-log DNA LADDER NEB 2μL + 4μL LB, 2) WELK Colony 1 PCR 5μL + 4μL LB, 3) WELK Colony 2 PCR 5μL + 4μL LB, 4) WELK Colony 3 PCR 5μL + 4μL LB, 5) WELK Colony 4 PCR 5μL + 4μL LB, 6) WELK Colony 5 PCR 5μL + 4μL LB, 7) WELK Colony 6 PCR 5μL + 4μL LB, 8) WELK Colony 7 PCR 5μL + 4μL LB,9)WELK Colony 8 PCR 5μL + 4μL LB, 10) WELK Colony 1 PCR 5μL + 4μL LB, 11) Negative control, 12)2-log DNA LADDER NEB 2μL + 4μL LB.
Fig 3. 1)2-log DNA LADDER NEB 2μL + 4μL LB, 2) WELK Colony 7 PCR restriction (X+P) 5μL + 4μL LB, 3) WELK Colony 9 PCR restriction (X+P) 5μL + 4μL LB, 4) WELK Colony 1 PCR restriction (X+P) 5μL + 4μL LB.
The documentation of the parts of our project, specifically BBa_K1904003 was made through Colony PCR, to validate the results of the colony PCR shown in fig 2 a digestion of the amplicon with (X+P) was made fig 3, where in the first well we can observe the expected weight of the insert (aprox 830 bp), therefore confirming the presence of our biobrick.
