Homologous Recombination of E2050 into pRS415 Construct in Place of GFP Protein

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<h1>Testing Bio-brick E2050 Part 1 - Homologous Recombination of  E2050 into GAL1p-[Npep-GFP] Construct in Place of GFP Protein</h1>
<h3>Aim</h3>
<p>The aim of this experiment was to homologously recombine the DNA sequence of Bio-brick E2050 mOrange (E2050), in place of green fluorescent protein (GFP), in GAL1p-[Npep-GFP] by transforming with yeast BY4741ΔTrp. This allowed testing of E2050 in further experiments.</p>
<h3>Hypothesis</h3>
<p>Since GFP has been detected when GAL1p-[Npep-GFP] is induced with galactose, if the E2050 is inserted in place of GFP in GAL1p-[Npep-GFP], then orange fluorescence should be detected under similar conditions for GFP expression.  Homologous recombination carried out by yeast is one technique that can be used to conveniently replace the GFP sequence with E2050 in GAL1p-[Npep-GFP]. <br>
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<h3>Protocol</h3>
<p>The Bio-brick E2050 was rescued from the DNA Distribution Kit provided by iGEM by transforming into sub-cloning DH5α E-coli strain. The plasmids were then extracted using a Qiagen Mini-Prep kit following manufacturer’s protocol. Testing for correct plasmid rescue was done by cutting extracted DNA with restriction enzymes XbaI and SpeI which are unique cut sites required to remove the DNA sequence that codes for mOrange from the bio-brick plasmid pSB2K3. Verification of correct sequence was by checking the length of insert and vector by gel electrophoresis of restriction digest. The expected fragment lengths are 744bp and 4425bp respectively.</p> <br>

<p>To homologously recombine the E2050 mOrange insert into GAL1p-[Npep-GFP] in place of GFP, the mOrange inserts were amplified by PCR using primers designed with 45bp overhangs that were homologous to the region of plasmid immediately prior and after the GFP sequence in GAL1p-[Npep-GFP]. The sequence to be amplified was checked to ensure that mOrange would be translated in-frame when integrated into GAL1p-[Npep-GFP].</p>  <br>

<p>In parallel, GAL1p-[Npep-GFP] constructs were restriction cut at unique sites using NheI and SmaI to remove the GFP coding sequence. Both digests were checked by gel electrophoresis to ensure the correct length of PCR product and cut vector was obtained (739bp and 965bp respectively).</p>  <br>

<p>For the homologous recombination, this was done by transforming yeast BY4741 auxotrophic for methionine, leucine, histidine, uracil and tryptophan with mOrange PCR products and cut GAL1p-[Npep-GFP] vector from previously. These would be homologously recombined by the yeast. The resultant transformants were selected by growing on SD agar with 1% each of methionine, histidine, uracil and tryptophan added since GAL1p-[Npep-GFP] has a selection marker for leucine encoded which is left intact throughout the experiment.</p> <br>

<p>To confirm that homologously recombined plasmids were selectively cultured, PCR colony screening was carried out on selected colonies from transformation plates.</p> <br>
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<h3>Results</h3>
<p>From (Table 1.) the results of the selective culture of transformants show that the positive and negative controls worked as expected with BY4741 growing when all nutrients are present and not growing in the absence of essential amino acids. The expected transformants also grew on selection plates 1 & 2 at the three volumes plated (50µl, 100 µl and 200 µl) with increasing density of colonies.</P> <br>

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<p>Since a transformation with ‘cut GAL1p-[Npep-GFP] only’ was not carried out, no information about the likelihood of uncut GAL1p-[Npep-GFP] from the restriction digest being transformed was obtained. To overcome this, individual colonies were selected and plated out for culture. PCR colony screening was then used to check that these had the mOrange insert transformed. Twenty colonies were selected, cultured and two colonies were PCR screened using primers for mOrange. (i.e. The primers used previously to generate mOrange with 45bp overhangs)  The results are shown by (Table 2.) and confirm that homologous recombination of mOrange PCR products had been successfully integrated into cut GAL1p-[Npep-GFP]. 
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<h3>Conclusions</h3>
<p>From the discussion of the results it has been shown that E2050 mOrange has been successfully rescued and amplified. These were then used as substrate for PCR amplification to generate product with homologous ends that were homologously recombined with cut GAL1p-[Npep-GFP], (GFP sequence removed) in a transformation process using BY4741ΔTrp. PCR colony screening using mOrange primers confirmed that colonies carrying the recombinant GAL1p-[Npep-GFP] had been successfully transformed into BY4741. 
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<h3>References</h3>
<p>Hinnen A, Hicks JB and Fink GR. Transformation of yeast.
Proc Natl Acad Sci, 75 1978  (1929-33)</p>

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