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iGEM CUHK

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Laccase Activity Test

25-09-2013

Detection the conversion of the o-phenylenediamine (ELISA substrate) to colour product by laccase

IPTG

Addition at OD=0.5

12h incubation

Laccase biobrick	Absent	Present	Present	Present	Present	Present	Present
IPTG (mM)	0	0	0.01	0.03	0.1	0.3	1
OD492nm	0.0662	0.0691	0.0722	0.0735	0.0771	0.0999	0.0968

	Wavelength	400	405	410	415	420	425	430	435	440	445	450	455
Laccase biobrick	IPTG (mM)	400nm	405nm	410nm	415nm	420nm	425nm	430nm	435nm	440nm	445nm	450nm	455nm
Absent	0	0.0778	0.076	0.0744	0.0736	0.0719	0.0713	0.0703	0.0694	0.0686	0.0686	0.068	0.0675
Present	0	0.0767	0.0751	0.0737	0.0725	0.0712	0.0706	0.0699	0.0692	0.0682	0.0687	0.0681	0.0683
Present	0.01	0.0791	0.0773	0.0761	0.0751	0.0742	0.0731	0.0728	0.0721	0.071	0.0716	0.071	0.0714
Present	0.03	0.0743	0.0728	0.0711	0.07	0.0693	0.0688	0.0681	0.0677	0.0675	0.068	0.0681	0.0692
Present	0.1	0.0761	0.0743	0.0727	0.072	0.0703	0.0699	0.0689	0.0685	0.0684	0.069	0.07	0.0711
Present	0.3	0.0964	0.0946	0.0931	0.0922	0.0909	0.0903	0.09	0.0901	0.0902	0.0917	0.0926	0.0943
Present	1	0.0947	0.0927	0.0912	0.0901	0.0887	0.0881	0.0879	0.0873	0.0878	0.0891	0.0898	0.0916

	Wavelength	460	465	470	475	480	485	490	495	500	505	510	515
Laccase biobrick	IPTG (mM)	460nm	465nm	470nm	475nm	480nm	485nm	490nm	495nm	500nm	505nm	510nm	515nm
Absent	0	0.0673	0.067	0.0667	0.0667	0.0667	0.0657	0.0652	0.0656	0.0646	0.0637	0.0629	0.0623
Present	0	0.0681	0.0677	0.0677	0.0675	0.0675	0.0674	0.068	0.0684	0.0672	0.0658	0.0635	0.0625
Present	0.01	0.0712	0.0711	0.0707	0.0709	0.0716	0.0709	0.0711	0.0715	0.0699	0.068	0.0658	0.065
Present	0.03	0.0696	0.0698	0.0703	0.0705	0.0712	0.0721	0.0726	0.0735	0.0701	0.0665	0.0621	0.0602
Present	0.1	0.072	0.0724	0.0731	0.0735	0.0749	0.0758	0.0767	0.0773	0.0732	0.0686	0.0631	0.0606
Present	0.3	0.0962	0.0971	0.0974	0.0983	0.1002	0.1029	0.1046	0.1054	0.0987	0.0922	0.0838	0.08
Present	1	0.0932	0.0937	0.0943	0.0951	0.0973	0.099	0.1004	0.101	0.0954	0.0893	0.0815	0.0774

	Wavelength	520	525	530	535	540	545	550	555	560	565	570	575
Laccase biobrick	IPTG (mM)	520nm	525nm	530nm	535nm	540nm	545nm	550nm	555nm	560nm	565nm	570nm	575nm
Absent	0	0.0615	0.0617	0.0616	0.0613	0.0607	0.0606	0.0607	0.0606	0.0602	0.0599	0.0596	0.0591
Present	0	0.0617	0.0611	0.061	0.0605	0.0595	0.0596	0.0593	0.0593	0.0591	0.0582	0.0579	0.0575
Present	0.01	0.0641	0.064	0.0636	0.0628	0.0622	0.0621	0.0615	0.0616	0.0614	0.0608	0.0607	0.06
Present	0.03	0.0586	0.0579	0.0573	0.0564	0.0559	0.0555	0.055	0.0552	0.0547	0.0543	0.0539	0.0537
Present	0.1	0.0585	0.0577	0.0572	0.0562	0.0553	0.055	0.0546	0.0541	0.0535	0.0531	0.0527	0.0525
Present	0.3	0.077	0.076	0.0744	0.0729	0.0723	0.0713	0.0707	0.0703	0.07	0.0694	0.0686	0.0685
Present	1	0.0749	0.0738	0.073	0.0712	0.07	0.0698	0.0693	0.069	0.0687	0.0682	0.0681	0.0678

	Wavelength	580	585	590	595	600
Laccase biobrick	IPTG (mM)	580nm	585nm	590nm	595nm	600nm
Absent	0	0.0584	0.0585	0.0582	0.0577	0.0579
Present	0	0.0573	0.0571	0.057	0.0564	0.0565
Present	0.01	0.0595	0.0594	0.0595	0.0589	0.0591
Present	0.03	0.0533	0.0532	0.053	0.0522	0.0528
Present	0.1	0.0524	0.052	0.0512	0.0512	0.0509
Present	0.3	0.0678	0.0675	0.0671	0.0668	0.0663
Present	1	0.067	0.0665	0.0665	0.066	0.0659

	Calculate the activity of laccase from acitivty of peroxidase
	1U is defined as μmol conversion of products per minute
Control	0	0.05	0.1	1	5
Peroxidase(mU)	0.0609	0.0744	0.0868	0.2405	0.4129

E. coli BL21 transformed with catechol 1,2-dioxygenase

wild type E. coli BL21

0        50.00           50.00
1        16.54           50.00
2        0.00 37.69

graph

Pearson's Chi-squared test

data: a
X-squared = 33.8347, df = 2, p-value = 4.497e-08

Cell Viability Experiment with Voltage

This experiment is designed to investigate the BL21 viability when voltage is applied. This is necessary for the voltage sensor experiments in our project. The protocol of the following experiment can be found here.(hyperlink to https://2013.igem.org/Team:Hong_Kong_CUHK/protocol)

Macintosh HD:Users:melisajunata:Desktop:Cell Viability Experiment with Voltage.png

Macintosh HD:Users:melisajunata:Desktop:Untitled.jpg

The difference in optical density (OD) before and after experiment determines the number of cells that grow during the experimental period.

The results show that the control group, BL21 E.coli without pSB1C3+RFP transformation survive better when voltage is applied as they are shaken in the incubator (25°C). On the other hand, the experimental group, BL212 E.coli with pSB1C3 + RFP shows a lower survival rate when voltage is applied and shaken inside the incubator (25°C).

Despite of the voltage being positive or negative, both groups show higher survival rate when lower magnitude voltage is applied, as shown on the graph.

Team:Hong Kong CUHK/results

From 2013.igem.org

Results

Laccase Activity Test

E. coli BL21 transformed with catechol 1,2-dioxygenase

Cell Viability Experiment with Voltage