Revision as of 10:03, 20 September 2015

Pcons+B0034+Lpp-OmpA-N+scFv(Anti-EGFR)+B0034+amilCP+B0015

2. Transformation of single plasmid

Introduction

To observe that our scFv can bind onto the cancer cells, we connected each scFv with different chromoprotein. Color proteins are commonly used as reporter gene for observation. However, chromoproteins can be conveniently observed by naked eye without the help of instruments. Therefore, when we conducted cell staining experiment, we can observe the binding distribution by color right after the experiments have finished.

(1) Parts:

Fig.13 Transformation of single plasmid

Fig.14 Pcons+RBS+Lpp-OmpA-N+Anti-EGFR+RBS+RFP+Ter

Fig.15 Pcons+RBS+Lpp-OmpA-N+Anti-EGFR+RBS+GFP+Ter

(2) Cell staining experiment: After creating the part of scFv and transforming them into our E. coli, we were going to prove that our detectors have successfully displayed scFv of anti-EGFR. To prove this, we have decided to undergo the cell staining experiment by using our E. coli to detect the EGFR in the SKOV-3 cancer cell lines. SKOV-3 is a kind of epithelial cell that expressed markers such as EGFR.

(3) Staining results：

Fig.16 As results,there is no red fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli.

Fig.17 There are green fluorescent anti-EGFR E. coli stick on the cell’s surface as the anti-EGFR probes on E. coli successfully detect and bind with EGFR.

Fig.18 As results,there is no green fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli.

Fig.19 There are green fluorescent anti-EGFR E. coli stick on the cell’s surface as the anti-EGFR probes on E. coli successfully detect and bind with EGFR.

Fig.20 As results,there is no green fluorescent E. coli stick on the cell’s surface as there is no specific scFv displayed around the E. coli.

Fig.21 There are blue chromoprotein anti-EGFR E. coli stick on the cell’s surface as the anti-EGFR probes on E. coli successfully detect and bind with EGFR.

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
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 451
1000
COMPATIBLE WITH RFC[1000]

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 <p style="font-size:120%">'''2. Transformation of single plasmid'''</p>
+<h1>'''Introduction'''</h1>
-To prove that our scFv can actually bind on to the antigen on cancer cells, we connected each scFv with a different fluorescence protein. Therefore we could use fluorescence microscope to clearly observe if the ''E. coli'' has produced scFv proteins. Currently, we built three different scFv connected with their respectively fluorescence protein. When applied on cell staining, we can identify the antigen distribution on cancer cells by observing the fluorescence. Furthermore, if we use the three scFv simultaneously, we can also detect multiple markers.
+To observe that our scFv can bind onto the cancer cells, we connected each scFv with different chromoprotein. Color proteins are commonly used as reporter gene for observation. However, chromoproteins can be conveniently observed by naked eye without the help of instruments. Therefore, when we conducted cell staining experiment, we can observe the binding distribution by color right after the experiments have finished.
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Difference between revisions of "Part:BBa K1694054"

Revision as of 10:03, 20 September 2015

Introduction