Difference between revisions of "Part:BBa K1632020"

Latest revision as of 00:43, 19 September 2015

rbs_CmR(ssrA degradation tag)

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.) 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.)

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(Fig.3. and Fig.4.).

Fig. 3. The results of modeling

Fig. 4.Adding an ssrA degradation tag

Fig. 5. The cells which have BBa_K1632022 growth with Cm

Fig. 6. The cells which have BBa_K1632023 growth with Cm

In the experiment using the Pcon_lasR_TT_Plux_CmRssrA (BBa_K1632022) (Fig.5.) and Pcon_rhlR_TT_Plux_CmRssrA (BBa_K1632023) (Fig.6.), 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
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1632020 short</partinfo>
-At the first stage of wet experiment, we used the cells which have the plasmid which is Pcon_lasR_TT_Plux_rbs_cmR to characterize the function of rbs_cmR(<partinfo>BBa_K395160</partinfo> by iGEM10_Tokyo_Tech). But the cells showed active growth even in the absence of AHL (Fig. 1.The cells growth with Cm)
+<span style="margin-left: 10px;">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.) to characterize the function of rbs_''CmR''(<partinfo>BBa_K395160</partinfo> by iGEM10_Tokyo_Tech). But the cells showed active growth even in the absence of AHL (Fig. 2.)
-[[Image:Tokyo_Tech Pcon_rbs_rhlR_TT_Plux_rbs_cmR.png|thumb|center|550px|<b>Fig. 1.</b> The cells growth with Cm]]
-From the results of this experiment, initially designed circuits showed leaky expression of CmR. Therefore we suspected that there was a leakage in the promoter. So we constructed a new plasmid with an ssrA degradation tag, rbs_cmRssrA.
+[[Image:Tokyo_Tech Pcon_rhlR_TT_Plux_cmR.png|thumb|center|450px|<b>Fig. 1.</b>Our initially design of a part containing a previously existing part]]
+[[Image:Tokyo_Tech Pcon_rbs_rhlR_TT_Plux_rbs_cmR.png|thumb|center|550px|<b>Fig. 2.</b> The cells growth with Cm]]
+<span style="margin-left: 10px;">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.3. and Fig.4.).
+[[Image:Tokyo_Tech Pcon_rbs_lsaR_TT_Plux_rbs_cmRssrA modeling.png|thumb|center|500px|<b>Fig. 3.</b> The results of modeling]]
+[[Image:ssrA tag .png|thumb|center|500px|<b>Fig. 4.Adding an ssrA degradation tag</b>]]
+[[Image:Tokyo_Tech Pcon_rbs_lsaR_TT_Plux_rbs_cmRssrA.png|thumb|center|440px|<b>Fig. 5.</b> The cells which have BBa_K1632022 growth with Cm]]
+[[Image:Tokyo_Tech Pcon_rbs_rhlR_TT_Plux_rbs_cmRssrA.png|thumb|center|440px|<b>Fig. 6.</b> The cells which have BBa_K1632023 growth with Cm]]
+<span style="margin-left: 10px;">In the experiment using the Pcon_''lasR''_TT_Plux_''CmRssrA'' (<partinfo>BBa_K1632022</partinfo>) (Fig.5.) and Pcon_''rhlR''_TT_Plux_''CmRssrA'' (<partinfo>BBa_K1632023</partinfo>) (Fig.6.), we could not observe cell growth for cells that owned the ssrA-taged plasmid, in the absence of AHL<br>
+<span style="margin-left: 10px;">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]]
 <!-- Add more about the biology of this part here