Difference between revisions of "Part:BBa K2382001"

Revision as of 20:07, 26 October 2017

MSMEG_5998

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
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 428
1000
COMPATIBLE WITH RFC[1000]

Short Description

To degrade aflatoxin in intestine as protection. We have chosen an enzyme of F420H2-dependent reductases family from Mycobacterium smegmatis. which can reduce and degrade aflatoxin with the aid of coenzyme F420.

Expression results

IPTG induction

MSMEG_5998 ( sequence is from Australia) were synthesized by Allbio Life Co., Ltd and put into the standard backbone pSB1C3. First, we transformed plasmid into E. coli BL21 (DE3) strain to express our proteins. Then IPTG was used to induce the expression system, since all plasmids in our project had T7 promoter. We sonicated E. coli and did 9500 rpm and 13000 rpm centrifugation to remove the cell pellet and obtain the supernatant. To confirm the suitable concentration of cell supernatant, we ran SDS-PAGE electrophoresis and did coomassie brilliant blue staining. The results are demonstrated in figure one. After centrifuging for two times, we could find a high percentage of proteins in the cell supernatant (the 13000 Su group).

figure 1

Figure 1: Cell lysates in the process of two times centrifuge were analyzed by western blot. 9500 T meant the initial sample gotten after sonication; 9500 P and 13000 T meant the pellet and the supernatant gotten after 9500 rpm for 20 min; 13000 P and 13000 Su meant the pellet and the supernatant gotten after 13000 rpm for 20 min.

References

(1)Taylor, M.C., et al., Identification and characterization of two families of F420H2‐dependent reductases from Mycobacteria that catalyse aflatoxin degradation. Molecular microbiology, 2010. 78(3): p. 561-575.

(2)Lapalikar, G.V., et al., F420H2-dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the Actinomycetales. PLoS One, 2012. 7(2): p. e30114.

@@ Line 20: / Line 20: @@
 ===Expression results===
 =====IPTG induction=====
-MSMEG_5998 sequence were synthesized by Allbio Life Co., Ltd and put into
+MSMEG_5998 ( sequence is from Australia) were synthesized by Allbio Life Co., Ltd and put into
-the standard backbone pSB1C3. First, we transformed our plasmid from Australia and two of our own composition parts into E. coli BL21 (DE3)
+the standard backbone pSB1C3. First, we transformed plasmid into E. coli BL21 (DE3)
 strain to express our proteins. Then IPTG was used to induce the expression system,
 since all plasmids in our project had T7 promoter. We sonicated E. coli and did 9500 rpm
 and 13000 rpm centrifugation to remove the cell pellet and obtain the supernatant. To
 confirm the suitable concentration of cell supernatant, we ran SDS-PAGE electrophoresis
-and did coomassie brilliant blue staining. The results are demonstrated in the Fig. 1A to
+and did coomassie brilliant blue staining. The results are demonstrated in figure one. After centrifuging for two times, we could find a high percentage of proteins in the
-C. After centrifuging for two times, we could find a high percentage of proteins in the
 cell supernatant (the 13000 Su group).
-[[File:Astralian MSMEG5998.png|250px|thumb|left|figure one]]
+[[File:Astralian MSMEG5998.png|250px|thumb|left|figure 1]]
 <p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
 <p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
@@ Line 44: / Line 43: @@
 <p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
 <p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
+Figure 1: Cell lysates in the process of two times centrifuge were analyzed by western blot. 9500 T meant the initial sample gotten after sonication; 9500 P and 13000 T
+meant the pellet and the supernatant gotten after 9500 rpm for 20 min; 13000 P and 13000 Su meant
+the pellet and the supernatant gotten after 13000 rpm for 20 min.
 ===References===