|
|
| Line 1: |
Line 1: |
| | __NOTOC__ | | __NOTOC__ |
| − | <partinfo>BBa_K1033933 short</partinfo> | + | <partinfo> 2.1-Å Crystal Structure Of A Nonfluorescent Chromoprotein From Anemonia Sulcata." Journal of Biological Chemistry 280.4 (2005): 2401-2404. |
| − | | + | |
| − | This chromoprotein from the coral ''Anemonia sulcata'', asPink (also known as asCP or asFP595), naturally exhibits strong color when expressed. The protein has an absorption maximum at 572 nm giving it a pink/purple color visible to the naked eye. The strong color is readily observed in both LB or on agar plates after less than 24 hours of incubation. The protein asPink has significant sequence homologies with proteins in the GFP family.
| + | |
| − | | + | |
| − | '''Important: This part is not available in the registry yet, however, the same part is available from the registry with the standard RBS B0034: <partinfo>BBa_K1033927</partinfo>.'''
| + | |
| − | <br><br>
| + | |
| − | | + | |
| − | ===Usage and Biology===
| + | |
| − | This part is useful as a reporter.
| + | |
| − | | + | |
| − | ===Contribution===
| + | |
| − | <div>
| + | |
| − | Group: Linkoping_Sweden iGEM 2019
| + | |
| − | <br>
| + | |
| − | Author: Andreas Holmqvist, Leo Juhlin and Emmanuel Berlin
| + | |
| − | <br>
| + | |
| − | Summary: We studied the expression in of <I>E.coli</I> BL21(DE3) containing CBD-asPink (<partinfo>BBa_K3182100</partinfo>) protein with a constitutive promotor to see if the protein could be used for a pink/white screening. We also tested the bindning capacity of the CBD-asPink protein to a micro cellulose bandage and then studied the release mechanism.
| + | |
| − | <br>
| + | |
| − | Documentation:
| + | |
| − | <html>
| + | |
| − | | + | |
| − | The expression system used contained the following parts:
| + | |
| − | <ul>
| + | |
| − | <li> </html>the [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034] Ribosome binding site<html></li>
| + | |
| − | <li> </html>the [https://parts.igem.org/Part:BBa_J23110 BBa_J23110] Constitutive promotor<html></li>
| + | |
| − | </ul>
| + | |
| − | <br><br>
| + | |
| − | <b> Pink-white screening </b><br>
| + | |
| − | To visualize the absorbance of AsPink on a petri dish, BL21 (DE3) colonies containing the pUC19-pCons-AsPink part (<a href="https://parts.igem.org/Part:BBa_K3182100">BBa_K3182100</a>), which was used in the pink-white screening, were incubated for 16 hours in 37 °C. The pCons-AsPink construct was removed and (Magainin 2) and <a href="https://parts.igem.org/Part:BBa_K3182104 ">BBa_K3182104</a> (CHAP) was inserted into the vector. The pink colonies contain AsPink and indicate a religated pCons-AsPink. The white colonies indicate a successful ligation. Below (Figure 1) is a picture of the pink-white screening.
| + | |
| − | | + | |
| − | </html>
| + | |
| − | [[File:T--Linkoping_Sweden--asPinkcompilation.png|350px|thumb|left|<b>Figure 2. </b><u>A:</u> E. coli BL21 cells expressing the biobrick, incubated for 16 hours at 16°C at 80 rpm in 1L of LB-miller. <u>B:</u> Lysated (via sonication) BL21s which are expressing the biobrick. It is lysate from the culture above in figure1. Incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller. <u>C:</u>Centrifuged lysate of BL21 culture which express the biobrick. It is the same culture as figure 4 and 5. A pellet of non-lysated bacteria can be observed.]]<html>
| + | |
| − | | + | |
| − | </html>[[File:T--Linkoping_Sweden--pinkwhite156.jpeg|510px|thumb|right|<b>Figure 1.</b>
| + | |
| − | BL21 (DE3) bacteria used for pink-white screening. The white colonies indicate a successful ligation and the pink colonies is AsPink expressing bacteria which signals a false-positive ligation. ]]<html>
| + | |
| − | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
| + | |
| − | <br><br><br><br>
| + | |
| − | <b>CBD-asPink bindning capacity</b><br>
| + | |
| − | </html>[[File:T--Linkoping Sweden--aspink bunden.jpeg|250px|right|thumb|<b><I>Figure 3.</I></b> Lysate (via sonication) from <I>E. coli</I> BL21 was incubated with Epiprotect (microbial cellulose bandage) for 1h and washed thrice with 70% ethanol. ]]<html>
| + | |
| − | To test the bindning capacity of CBD-asPink (<partinfo>BBa_K3182000</partinfo>) the microbial cellulose bandage was suspended into sonicated lysate of <I>E.coli</I> BL21(DE3) with the expressed fusion protein (<b><I>Figure 3</I></b>) and incubated for 30 minutes.
| + | |
| − | The bandage was then washed thrice in 70% ethanol which confirmed that the bindning of CDB-asPink was specific for the cellulose bandage.
| + | |
| − | <br><br>
| + | |
| − | </html>[[File:T--Linkoping Sweden--aspink bunden vs obunden.jpeg|250px|left|thumb|<b><I>Figure 4.</I></b> To test the release mechanism of CBD-asPink, human thrombin and cleavage buffer was added to the bandage and incubated for 16 hours on an end to end rotator. The figure is the result after the incubation with a negative control with only cleavage buffer (left) and human thrombin and cleavage buffer (right). ]]<html> <b> CBD-asPink with thrombin cleavage</b><br>
| + | |
| − | | + | |
| − | After the washes, human thrombin and cleavage buffer was added to the bandage with bound CBD-asPink to test the release mechanism of the fusion protein. The bandage was then incubated with the solution for 16 hours on an end to end rotator together with an negative control containing cellulose bandage and only cleavage buffer.
| + | |
| − | After the incubation, the supernatant containing thrombin was pink while the negative control containing only cleavage buffer was transparent with a clear pink cellulose bandage (<b><I>Figure 4</I></b>). This indicates that the release mechanism work and the AsPink protein has successfully been released from the bandage into the supernatant.
| + | |
| − | <html>
| + | |
| − | | + | |
| − | <br><br>
| + | |
| − | </html>
| + | |
| − | | + | |
| − | <html>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | </html>
| + | |
| − | <html>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | </div>
| + | |
| − | </html>
| + | |
| − | <br><br><br><br><br><br>
| + | |
| − | | + | |
| − | | + | |
| − | asPink does not exist as CDS only but only as RBS-asPink (<partinfo>BBa_K1033927</partinfo>) and J23110-RBS-spisPink (<partinfo>BBa_K1033926</partinfo>).
| + | |
| − | | + | |
| − | [[Image:AsPink.jpg|700px]]
| + | |
| − | | + | |
| − | '''iGEM2013 Uppsala:''' Expression of asPink in ''E. coli'' DH5alpha by promoter J23110 from medium copy plasmid pSB3K3 (left) or high copy plasmid pSB1K3 (right).
| + | |
| − | | + | |
| − | ===Source===
| + | |
| − | The protein was first extracted and characterized by Lukyanov et. al. 2000 under the name asFP595 (GenBank: AAG02385.1). This version is codon optimized for '' E coli'' by Genscript.
| + | |
| − | | + | |
| − | ===References===
| + | |
| − | [http://www.jbc.org/content/275/34/25879.short]Lukyanov, Konstantin A., et al. "Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog." Journal of Biological Chemistry 275.34 (2000): 25879-25882.
| + | |
| − | | + | |
| − | [http://www.jbc.org/content/280/4/2401.short]Wilmann, Pascal G., et al. "Variations on the GFP Chromophore - A Polypeptide Fragmentation Within The Chromophore Revealed In The 2.1-Å Crystal Structure Of A Nonfluorescent Chromoprotein From Anemonia Sulcata." Journal of Biological Chemistry 280.4 (2005): 2401-2404.
| + | |
| | | | |
| | | | |
