Difference between revisions of "Part:BBa K103006:Experience"

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<I>Iowis Zhu</I>
 
<I>Iowis Zhu</I>
 
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BBa_K103006 is difficult to use when assembling a composite biobrick using RE cloning for the pSB1C3 vector backbone. The SacI site designed into the linker can also be found in the chloramphenicol acetyltransferase gene in pSB1C3. This increases the hassle of RE cloning as the backbone would also be cut along with the insert. BBa_K1489003 replaces the SacI site with a KasI site in order to avoid this issue.
 
BBa_K103006 is difficult to use when assembling a composite biobrick using RE cloning for the pSB1C3 vector backbone. The SacI site designed into the linker can also be found in the chloramphenicol acetyltransferase gene in pSB1C3. This increases the hassle of RE cloning as the backbone would also be cut along with the insert. BBa_K1489003 replaces the SacI site with a KasI site in order to avoid this issue.
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[https://parts.igem.org/Part:BBa_K1489003 BBa_K1489003]
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Latest revision as of 06:21, 26 October 2014

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K103006

User Reviews

UNIQde5c74beafade6c1-partinfo-00000000-QINU

•••••

Paweł Krawczyk

This part was used as a outer-membrane-targeting factor in 'hunter' part of our project.

We've checked if proteins fused to OmpA are present on the outer cell membrane of E.coli - please take a look on our [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=2&arg0=12_August_2008&arg1=13_August_2008&name=Checking%20for%20presence%20of%20protein%20A%20on%20the%20cell%20membrane notebook entries]

This part was used in fusion with another parts:

For detailed information please check 'Experience' subpages of those parts using links above.

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••••

Yunxiao

This part contains a fragment sufficient to transport the target protein outside the bacteria. We tested the expression conditions for optimum transport.

In our part

All used OmpA for efficient transport onto the outer membrane of E. coli.

In our part Part:BBa_K533005, we fused OmpA to GFP and inserted a his tag for convenient detection.

After induction at 18 centigrade for 12 hours in BL21, we harvested the bacteria and digested it with Protease K at 50 centigrade for 30min.

Color Channel Post-digest Pre-digest
GFP Thuexp gn.png Thuexp gp.png
DAPI Thuexp gn d.png Thuexp gp d.png
Merge Thuexp gn m.png Thuexp gp m.png

The results suggested that GFP was efficiently transported onto the outer membrane.

Cellular fractionation and western blotting confirmed the results that GFP is predominantly on the membrane.

Thuexp west.png

We further tested the induction conditions with our part Part:BBa_K533002.


Color Channel 0.1mM IPTG induction 0.5mM IPTG induction 1.0mM IPTG induction
mCherry Thuexp bind 0.1.png Thuexp bind 0.5.png Thuexp bind 1.0.png
DAPI Thuexp bind 0.1 d.png Thuexp bind 0.5 d.png Thuexp bind 1.0 d.png
merge Thuexp bind 0.1 m.png Thuexp bind 0.5 m.png Thuexp bind 1.0 m.png

It seems that the transport efficiency is highest when BL21 is induced at 0.1mM IPTG at 18 centigrade. This result suggests that, under the drive of an extremely strong promoter as T7, lowering expression level is necessary for efficient transportation.

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•••

Iowis Zhu

BBa_K103006 is difficult to use when assembling a composite biobrick using RE cloning for the pSB1C3 vector backbone. The SacI site designed into the linker can also be found in the chloramphenicol acetyltransferase gene in pSB1C3. This increases the hassle of RE cloning as the backbone would also be cut along with the insert. BBa_K1489003 replaces the SacI site with a KasI site in order to avoid this issue. BBa_K1489003

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UNIQde5c74beafade6c1-partinfo-00000004-QINU