Difference between revisions of "Part:BBa K3580003:Experience"

Revision as of 15:49, 27 October 2020

Applications of BBa_K3580003

In our project, we inserted this parts into pSB1C3 plasmid.

In order to compare these parts(BBa_K3580003 and BBa_K934025), we first measured the fluorescence of GFP expressed in vivo. Each part was transformed into an E. coli JM2.300 strain with luxR plasmid introduced into that. After overnight incubation, diluted and OD600-matched to fresh Culture and 3OC6HSL induction was added to the corresponding cultures of both parts at the appropriate OD600. After that, the OD600 of the culture was measured every 1h and the GFP fluorescence values in vivo were measured by diluting aliquot for measurement from the culture to a constant turbidity (OD600=0.4) at each measurement (Fig1). The measured GFP fluorescence values of the final time point (240 min) from the start of induction at each condition are shown in Fig 2. The fluorescence of tagged GFP (BBa_K3580003) was lower than that of normal GFP(BBa_K934025) at 240 min point (Fig1). Although GFP is a stable protein with a β-barrel and much difficult to be degraded, this result shows that tagged GFP was successfully degraded in vivo as we desired. 

Fig1 continuous measurement in vivo

Fig2 ssrA tag assay in vivo

Then, we compared the fluorescence of GFP in a cell-free system which was extracted from the E.coli containing luxR protein (Fig 3). Because of the programmed degradation, the fluorescence of tagged GFP(BBa_K3580003) showed slight signal nearly equal to a negative control where neither the template GFP DNA nor the inducer AHL exists. Those results show that ssrA tagged protein can be degraded much both in vivo and in vitro. Based on this data, we modified the degradation terms in the model, as described below.

Fig3 ssrA tag assay in vitro

User Reviews

UNIQ7f1c348a0d5839a5-partinfo-00000000-QINU UNIQ7f1c348a0d5839a5-partinfo-00000001-QINU

@@ Line 4: / Line 4: @@
 ===Applications of BBa_K3580003===
 In our project, we inserted this parts into pSB1C3 plasmid.
+In order to compare these parts(BBa_K3580003 and BBa_K934025), we first measured the fluorescence of GFP expressed in vivo.
+Each part was transformed into an E. coli JM2.300 strain with luxR plasmid introduced into that. After overnight incubation, diluted and OD600-matched to fresh Culture and 3OC6HSL induction was added to the corresponding cultures of both parts at the appropriate OD600. After that, the OD600 of the culture was measured every 1h and the GFP fluorescence values in vivo were measured by diluting aliquot for measurement from the culture to a constant turbidity (OD600=0.4) at each measurement (Fig1). The measured GFP fluorescence values of the final time point (240 min) from the start of induction at each condition are shown in Fig 2.
+The fluorescence of tagged GFP (BBa_K3580003) was lower than that of normal GFP(BBa_K934025) at 240 min point (Fig1). Although GFP is a stable protein with a β-barrel and much difficult to be degraded, this result shows that tagged GFP was successfully degraded in vivo as we desired.
+[[File:T--Waseda--degradation-continuous.png|500px|thumb|center|<b>Fig1</b> continuous measurement <i>in vivo</i>]]
+[[File:T--Waseda--Plux-tet-GFPssrA-vivo-comment revised.png|500px|thumb|center|<b>Fig2</b> ssrA tag assay <i>in vivo </i>]]
+Then, we compared the fluorescence of GFP in a cell-free system which was extracted from the E.coli containing luxR protein (Fig 3). Because of the programmed degradation, the fluorescence of tagged GFP(BBa_K3580003) showed slight signal nearly equal to a negative control where neither the template GFP DNA nor the inducer AHL exists. Those results show that ssrA tagged protein can be degraded much both in vivo and in vitro. Based on this data, we modified the degradation terms in the model, as described below.
+[[File:T--Waseda--Plux-tet-GFPssrA-vitro-comment.png|500px|thumb|center|<b>Fig3</b> ssrA tag assay <i>in vitro </i>]]
 ===User Reviews===