Generator

Part:BBa_K741002

Designed by: Wuyang Chen   Group: iGEM12_USTC-China   (2012-07-24)

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.

USTC2012Result1 副本.png

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


                                                         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
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