Part:BBa_K741002

plac-RBS-GFP-T

Lac promoter with GFP gene downstream. LacI or glucose can repress the expression of GFP while lactose and IPTG can activates it.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 870
  

Initially, we use IPTG to activate the promoter plac. However, we find that the leak expression of plac is quite high. Even without the existence of IPTG, GFP expresses. This is because there are no lacI genes on the plasmid bones PSB1C3. And K12 strains can not express enough lacI to repress the expression of plac. We look into some documents and find that glucose can repress the expression of plac(see the character of the plac in the lactose operon).Therefore, we set four experimental groups, adding glucose with concentration of 500mM, 800mM, 1000mM, 1000mM of IPTG and a blank group with nothing added.

From figure 1, we can see the unit fluorescence intensity of experimental groups with glucose added is lower than the blank group, because glucose represses the expression of plac, which reduces the expression of GFP.

At the beginning, we can’t observe the activating effect of IPTG on plac, which we ascribe to the unstability of the promoter plac. About 90 minutes later, the unit fluorescence intensity of experimental groups with IPTG added is higher than the blank group, which indicates IPTG’s activation on plac.

The difference of the unit fluorescence intensity between the experimental groups with different concentrations of glucose added is not strictly related to the increasing amount of glucose, because glucose affects the growth and metabolism of E.coli, which brings uncertainty to our experiments.

About 3 hours later, the difference of the unit fluorescence intensity between the groups abates, because of the consumption of glucose.

Data

Fluorescence intensity
plac-RBS-GFP-T
t/min	M9 medium	0			500mM glucose			800mM glucose			1000mM glucose			1000mM IPTG
Parallel	1	1	2	3	1	2	3	1	2	3	1	2	3	1	2	3
0	11.801	197.047	198.876	190.953	202.614	203.782	202.702	200.39	202.035	204.525	206.516	206.355	206.741	179.988	210.315	205.464
45	13.386	214.145	213.602	219.416	223.437	241.267	224.909	244.257	235.427	243.904	224.783	235.979	239.014	209.009	214.669	224.21
90	11.541	273.664	245.629	250.211	305.733	317.547	313.495	299.869	324.396	328.157	331.194	309.167	305.24	261.801	267.369	281.328
135	11.894	344.987	319.854	325.421	370.167	398.352	369.309	372.487	407.478	385.073	333.989	355.2	351.929	314.246	332.2	351.364
180	11.066	424.182	425.564	389.785	415.844	463.184	378.606	447.508	456.27	449.414	311.226	441.902	456.969	338.101	304.146	349.339

Functional Parameters

OD
plac-RBS-GFP-T
t/min	M9 medium	0			500mM glucose			800mM glucose			1000mM glucose			1000mM IPTG
0	0.045	0.0726	0.0719	0.0733	0.084	0.0829	0.0754	0.0747	0.0807	0.0741	0.0785	0.0883	0.0863	0.075	0.073	0.0781
45	0.0436	0.0793	0.0792	0.0798	0.0932	0.0743	0.0861	0.0834	0.0909	0.086	0.0873	0.1011	0.098	0.0835	0.0813	0.0875
90	0.0435	0.0973	0.0978	0.1004	0.1231	0.1222	0.1165	0.1165	0.1172	0.1166	0.1147	0.131	0.1255	0.0978	0.0974	0.104
135	0.0447	0.1403	0.1367	0.135	0.1611	0.1551	0.1625	0.1601	0.1668	0.1609	0.1852	0.1787	0.1755	0.1463	0.1349	0.1218
180	0.0446	0.1995	0.2064	0.2297	0.2414	0.2346	0.2161	0.239	0.2405	0.2209	0.1884	0.2627	0.23	0.1832	0.2	0.1967