Difference between revisions of "Part:BBa K1906005"

Revision as of 18:05, 20 October 2016

Beta-subunit of Qbeta replicase holoenzyme
This protein is the beta subunit of Qbeta replicase.

Characterlization

It was quite surprising that plasmids that carry beta subunit genes don't lead to a heavy production of the protein upon the induction. However, this situation can be improved when EF-Ts and EF-Tu are co-expressed with beta subunit.

Figure 1 is the SDS-PAGE result comparison between cells that transformed with these three proteins and beta subunit alone.

Figure 1. SDS-PAGE result comparison between three-protein-expressing cells and beta subunit-expressing cells, wild-type cells were included as a control group. The gel on the left showing the results of cells that been induced with 2mM IPTG and the gel on the right shows the results of same groups of cells that not been induced. The blue arrow indicates the band position of beta subunit.

It can be clearly seen that cells transformed with beta subunit alone had no detectable level of protein expression. However, the expression of beta subunit can be greatly restored when plasmids carry EF-Tu and EF-Ts are transformed into the cells.

Usage and Biology

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1317
Illegal BamHI site found at 1460
Illegal XhoI site found at 991
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1473
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 ===Characterlization===
-Whole cell lysate, supernatant and precipitations of cells with different constructs are loaded into SDS-PAGE gels. Figure 3 shows the electrophoresis results of samples that transformed with three different constructs: 1. pETDuet-1-Rep (Expressing β subunit alone)	2. pACYC-TS-TU (co-expressing EF-Ts and EF-Tu) 3. pACYC-TU (Expressing EF-Tu alone) It can be clearly observed that, unpon inducement of IPTG, pETDuet-1-Rep doesn't lead to the abundant production of β subunit. None of the three lanes of cells that transformed with pETDuet-1-Rep display a detectable band at 65.6 kDa, the size of β subunit. The cells that transformed with pACYC-TS-TU and pACYC-TU, however, shows unambiguous production of EF-Ts (position indicated by red arrow) and EF-Tu (position indicated by orange arrow).
-[[File:20161019150339!SDS-TSTU-REP.jpg|200px|thumb|left|alt text]]
+<P>It was quite surprising that plasmids that carry beta subunit genes don't lead to a heavy production of the protein upon the induction. However, this situation can be improved when EF-Ts and EF-Tu are co-expressed with beta subunit.</P>
-[[File:SDS-TSTU-REP.jpg|200px|thumb|left|alt text]]
+<p>Figure 1 is the SDS-PAGE result comparison between cells that transformed with these three proteins and beta subunit alone.</P>
+[[File:20161019150339!SDS-TSTU-REP.jpg|500px|thumb|left|Figure 1. SDS-PAGE result comparison between three-protein-expressing cells and beta subunit-expressing cells, wild-type cells were included as a control group. The gel on the left showing the results of cells that been induced with 2mM IPTG and the gel on the right shows the results of same groups of cells that not been induced. The blue arrow indicates the band position of beta subunit.]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+It can be clearly seen that cells transformed with beta subunit alone had no detectable level of protein expression. However, the expression of beta subunit can be greatly restored when plasmids carry EF-Tu and EF-Ts are transformed into the cells.
 ===Usage and Biology===