Part:BBa_K523015:Experience

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Team TecCEM_HS 2016

The TecCEM_HS decided to further work with the part BBa_K523015, which was characterized via a DNS assay. It consists of Lac promoter RBS and endoglucanase from C. Fimi. Optimum temperature was determined.

Two liters of DNS were prepared with 20 g of NaOH, 4 g of phenol, 1 g of sodium sulfite, 400 g of potassium sodium tartrate and 20 g of DNS. Carboxymethylcellulose (CMC) was used as substrate and all assays were realized with CMC 2% in PBS pH 7. All assays, including the standard curve were done triplicate.

A standard curve of glucose was realized ranging from 0.1 to 2 mM from a 10 mM stock and diluted with PBS pH 7. In order to reveal the reaction DNS was added and absorbance at 540 nm was determined. Finally data was analyzed in a spreadsheet, plotted obtaining an equation (y=0.8709x-0.1124) with a correlation factor equal to 0.9285

Figure 1 Standard Glucose-DNS Curve.

The biobrick was transformed in E. coli strain BL21 in order to express the enzyme endoglucanase, and positive clones were selected by antibiotic resistance. As reported before in literature, endoglucanese is an extracellular enzyme but in order to have more activity, lysis of cells is recommended. Positive clones were further grown overnight in LB liquid media with proper antibiotic (chloramphenicol-CAM) concentration. The first step consisted on centrifugation at 10,000 g for 15 minutes to eliminate growth medium, after resuspension in 10 mL of miliQ water, lysis of cells was achieved with an homogenizer “Polytron PT2100”, finally another centrifugation to collect all non soluble parts of cells and supernatant was used.

The assays were incubated at 20 °C, 25°C, 30°C, 35°C, 37°C and 40°C, based on literature. In a microtube 300 μL of CMC 2% was added, followed by 150 μL of supernatant of lysis and the mix was incubated 30 minutes at selected temperature. To stop and reveal the reaction 600 μL of DNS were added and absorbance at 540 nm was determined. In order to obtain the optimum temperature the average of each temperature was transformed to concentration of glucose hydrolyzed with the standard curve, furthermore μmol of substrate hydrolyzed was obtained by multiplying the concentration of glucose and the volume of substrate added. At last, activity was calculated dividing μmol of substrate between time of assay. Results are shown in table and plotted as a function of temperature. As seen in the graphic of temperature against activity, 30 °C and 37°C can be concluded as the optimal temperatures due to a higher activity but in table it can be discerned that 30°C presented higher activity.

Temperature	Absorbance average	μmol of substrate	Activity (UI)
20 °C	0.029	0.0489	0.00163
25 °C	0.069	0.0624	0.00208
30 °C	0.174	0.0988	0.00329
35 °C	0.060	0.0596	0.00198
37 °C	0.082	0.0671	0.00223
40 °C	0.171	0.0978	0.00326

Further Characterization of BBa K523015 on S-layer of Caulobacter crescentus

Contributed by British_Columbia 2016 iGEM team.

We have codon-optimized cenA for the expression in Caulobacter crescentus and synthesized the part by IDT(Submitted to Registry as BBa_K2139017). We have cloned the region from 190 to 447 aa position into p4A_723 rsaA plasmid to be displayed on the surface of C.crescentus. We performed as assay with DNP-cellobiose substrate, which consists of cellubiose bound to a chromophore, to test activity of the cenA expressed on cell surface. The test is specific to cellulolytic activities and the activity can be measured as absorbance 400. The 150 μL of Caulobacter crescentus cultures expressing cenA and wild-type rsaA were incubated with 150 μL of assay mix (0.1 mg/ml DNPC in 50 mM pH 5.5 potassium acetate buffer) and the absorbance at 400 and 600 nm was measured over 12 hours. Unfortunately, cenA hasn't shown activity on the surface layer of Caulobacter crescentus.

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