Part:BBa_K1632022

In our project, we wanted to use the CmR. At the first stage of wet experiment, we used the cells which have the plasmid which is Pcon_rhlR_TT_Plux_rbs_CmR to characterize the function of rbs_CmR(BBa_K395160 by iGEM10_Tokyo_Tech). But the cells showed active growth even in the absence of AHL (Fig. 1.The cells growth with Cm)

Fig. 1. The cells growth with Cm

From the results of this experiment, initially designed circuits showed leaky expression of CmR. We came up with two solutions, either increasing the chloramphenicol (Cm) concentration or inserting an ssrA tag to the CmR gene, to this problem. From modeling allowed us to successfully solve the influence of the leakage by adding an ssrA degradation tag right after the CmR gene (Fig. 2.The results of modeling).

Fig. 2. The results of modeling

Fig. 3. The cells which have BBa_K1632022 growth with Cm

In the experiment using the Pcon_lasR_TT_Plux_CmRssrA (BBa_K1632022) (Fig.3 The cells which have BBa_K1632022 growth with Cm), we could not observe cell growth for cells that owned the ssrA-taged plasmid, in the absence of AHL
From our experiment, CmRssrA is confirmed work better than CmR without ssrA tag for our project.

More information

For more information, see http://2015.igem.org/Team:Tokyo_Tech/Project Our work in Tokyo_Tech 2015 wiki, http://2015.igem.org/Team:Tokyo_Tech/Experiment/ssrA_tag_degradation_assay About ssrA-tag, http://2015.igem.org/Team:Tokyo_Tech/Experiment/Overview_of_fim_inversion_system About ''fim'' inversion system

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 383
  Illegal AgeI site found at 580
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 927