Team:Gothenburg-Sweden/Lab Note

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Chalmers University of Technology

preliminary work & lab notes

preliminary

Fluorescent Proteins:

Bioluminescence is used as a defense, offense and communication mechanism by various organisms in nature including insects, fish, squid, sea cacti, sea pansies, clam, shrimps and jellyfish(1). After the discovery of the Green Fluorescent Protein (GFP) by Shimomura et al. from Aequorea jellyfish(2), an unprecedented tool for visualizing living organisms was also being introduced to molecular biology, leading to a Nobel Prize in 2008(3). The Aequorea GFP is a 238 aa, 30 kDa monomer that emits green fluorescence maximum at 509 nm wavelength(4). It has a unique structure with an 11 stranded β - barrel like shape involving α- helixes running through the center of the can; to which the chromophore is attached(5). Autocatalytic formation of the chromophore of the GFP without the neccessity of any substrates or cofactors is a rather useful property of the GFPs allowing them to express in fusion with different proteins. Remarkably, the fusion of GFP to a protein does not have any vital effect on the activity or mobility of the protein in addition to its nontoxic nature(1).

GFP

Figure 1: Structure of the GFP taken from the PDB, code 1EMA

References:

(1) Zimmer, M. (2002) “Green Fluorescent Protein (GFP): Applications, Structure and Related Photophysical Behavior.” Chemical Reviews. v. 102 (3) pp. 759-781.
(2) Shimomura, O., Johnson, F.H. & Saiga, Y. (1962) “Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan.” Aequorea. J. Cell. Comp. Physiol. v. 59, pp. 223–239.
(3) Shimomura, O., Chalfie, M. & Tsien, R. Y. (2008) “The discovery and development of the green fluorescent protein, GFP.” The Nobel Prize in Chemistry. Retrieved from the website http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/
(4) Prasher, D., C., Eckenrode, V., K., Ward,W.,W., Prendergast, F., G. & Cormier, M., J. (1992) “Primary structure of the Aequorea victoria green-fluorescent protein” Gene., v. 111, pp. 229-233.
(5) Tsien, R., Y. (1998) “The Green Fluorescent Protein” Annual Review of Biochemistry. v. 67, pp. 509–544

Plasmid backbone:

pSP-GM1

SNF1 (modified subunits):

Snf1 (alfa), Snf4 (gamma)

FP positions:

N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma

Primer design:

Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C

lab work by date

2010-07-05
Malin och Katarina
An overnight culture were made from 4 different colonies of E.coli containing the plasmid pSB-GM1.

2010-07-06
Malin, Katarina and Peidi
Colony 1 and 2 were picked to do a miniprep. The plasmids were extracted and a plasmid integrity check was performed. By cutting with BamHI, one cut were made to get the size of the plasmid. Then we cut with ClaI which is a two-cutter that gives a fragment that is 600 bp and one that is 6800. The gel which should confirm this was not satisfactory since the 600 band was missing.

2010-07-07
Adnan, Katarina and Julia
As the gels from Tuesday showed ambigous results we decided to redo the miniprep of the plamids. We used the overnight preparations of the two unused colonies (3 & 4). The miniprep went smoothly except for major difficulties regarding untrustworthy pipettes. The pipettes marked with green tape are the ones to use from now on together with the tips with the same markings! A gel was molded and this time we decided to use a full gel to get a better resolution. The results on the gel where very good. The plasmids had clearly been cut in one or two places and the fragment sizes seem to correspond to the expected sizes.

2010-07-08
Malin, Julia and Peidi
The two BioBricks EYFP and ECFP were extracted from the kit plates and stored in the freezer. A SOC-medium was prepared.

2010-07-09
Malin and Julia
The SOC medium was autoclaved. Competent E.coli cells were transformed with our FPs through heat shock and plated on petri dishes. 20 new LB-amp plates were made.
Adnan and Katarina
Picked up primers at Chalmers and checked if they were ok. The concentration of the genomic DNA (SNF1 gene) were measured. Prepared a master mix so that the PCR-reactions could be started Monday morning.

2010-07-13
Karl and Adnan
We made duplicates of the haploid Snf4∆ and wildtype strains on new plates in case the original plate would be contaminated. Prepared a sporulation medium that was autoclaved and poured into empty growth plates to stay in room temperature over night.
Julia and Lokesh
We run the gel with products of our first three PCRs along with the templates we made for the two fluorescent proteins. We also prepared the sample solutions of DNA templates with appropriate concentration(1ng/µl) for the next PCR reactions.

pictures

PIC 1: Gel of plasmid (cut)

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                                     N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma
 				    <p>&nbsp;</p>
-				    <p class="STYLE6">Primers  design:</p>
+				    <p class="STYLE6">Primer  design:</p>
-                                    Fusion primers with restiriction enzyme sites included at very ends
+                                    Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C
 				    <p>&nbsp;</p></td>
 		  </tr>
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 						        <table width="541" border="1">
                                    <tr>
-                                     <td>PIC 1: Gel of plasmid (cutted) </td>
+                                     <td>PIC 1: Gel of plasmid (cut) </td>
                                    </tr>
                                  </table></td>