Part:BBa_K1632023

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(Fig. 1. Our initially design of a part containing a previously existing part) 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. 2.The cells growth with Cm)

Fig. 1.Our initially design of a part containing a previously existing part

Fig. 2. 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.

File:Tokyo Tech Pcon rbs lsaR TT Plux rbs cmRssrA modeling.png .png

Fig. 3. The results of modeling

Fig. 4. The cells which have BBa_K1632023 growth with Cm

In the experiment using the Pcon_ Pcon_rhlR_TT_Plux_CmRssrA (BBa_K1632023) (Fig.3 The cells which have BBa_K1632023 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.

For more information, see [http://2015.igem.org/Team:Tokyo_Tech/Project our work in Tokyo_Tech 2015 wiki].

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 301
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 776
  Illegal BsaI.rc site found at 933