Difference between revisions of "Team:Humboldt Berlin/Improve"

 
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                                     <a href="/Team:Humboldt_Berlin/Parts">Parts
 
                                     <a href="/Team:Humboldt_Berlin/Parts">Parts
 
                                         overview</a>
 
                                         overview</a>
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                                    <a href="/Team:Humboldt_Berlin/Basic_Part">Basic
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                                        parts</a>
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                                <a href="/Team:Humboldt_Berlin/Basic_Part">Basic parts</a>
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                                    <a
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                                <a href="/Team:Humboldt_Berlin/Composite_Part">Composite parts</a>
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                                        href="/Team:Humboldt_Berlin/Composite_Part">Composite
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                                        parts</a>
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                                     <a
 
                                     <a
 
                                         href="/Team:Humboldt_Berlin/Part_Collection">Parts
 
                                         href="/Team:Humboldt_Berlin/Part_Collection">Parts
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                                     <a href="/Team:Humboldt_Berlin/Model">Model</a>
 
                                     <a href="/Team:Humboldt_Berlin/Model">Model</a>
 
                                     <a href="/Team:Humboldt_Berlin/Plant">Plant</a>
 
                                     <a href="/Team:Humboldt_Berlin/Plant">Plant</a>
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                                    <a href="/Team:Humboldt_Berlin/Software">Software</a>
 
 
                                 </div>
 
                                 </div>
 
                             </div>
 
                             </div>
 
                             <div class="devider"></div>
 
                             <div class="devider"></div>
 
                             <a
 
                             <a
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                                 href="https://igem.org/2019_Judging_Form?team=Humboldt_Berlin">
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                                 href="https://2019.igem.org/Team:Humboldt_Berlin/Achievements">
 
                                 For Judges
 
                                 For Judges
 
                             </a>
 
                             </a>
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             <!------------------------------------------ HEADER END -------------------------------------------------->
 
             <!------------------------------------------ HEADER END -------------------------------------------------->
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            <section class="page-content fixed-header-content width-limit">
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<div class="padding-container">
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<section class="page-content fixed-header-content width-limit">
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                <h3 class="headline3">Introduction</h3>
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                <p class="block-text medium-sized">
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    <!------------------------------------------ TWO COLUMN IMG RIGHT ------------------------------------------------>
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                    Paromomycin belongs to a group of aminoglycoside antibiotics
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                <div class="two-columns">
 +
                    <div>
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                        <h3 class="headline3">Introduction</h3>
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                        <p class="block-text medium-sized">
 +
                            Paromomycin belongs to a group of aminoglycoside antibiotics
 
                     such as neomycin or dibekacin. These aminoglycosides are
 
                     such as neomycin or dibekacin. These aminoglycosides are
 
                     capable of inhibiting the eukaryotic translation, by binding
 
                     capable of inhibiting the eukaryotic translation, by binding
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                     within the large and small subunit of the 80S ribosome. This
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                     within the large and small subunit of the 80S ribosome. The bacteria
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                    property allows paromomycin to be used as selection marker
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                     <i>Streptomyces rimosus</i> carries the aminoglycoside
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                     for <i>C. reinhardtii</i>. For the selection process to
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                     3’-phosphotransferase encoded in the so-called <i>aphVIII</i> gene.
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                    work, one must consider a way to implement a paromomycin
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                     This enzyme inhibits paromomycin by transferring the gamma-phosphate of
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                    resistance in <i>C. reinhardtii</i>. The bacteria
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                    Stretpomyces rimous carries the aminoglycoside
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                     3’-phosphotransferase encoded in the so called aphVIII gene.
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                     This enzyme catalyses the transfer of the gamma-phosphate of
+
 
                     ATP to the hydroxyl group in 3’ position of the paromomycin
 
                     ATP to the hydroxyl group in 3’ position of the paromomycin
 
                     molecule and allows the carrier of the gene to develop a
 
                     molecule and allows the carrier of the gene to develop a
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                     resistance to paromomycin (Sizova et al. 2001). We used this
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                     resistance to paromomycin. The <i>aphVIII</i> gene is a commonly used transformation marker  in green algae and established for the use in <i>C. reinhardtii</i> (Sizova et al. 2001) and was submitted to the registry last year as <a href="http://parts.igem.org/Part:BBa_K2703008">BBa_K2703008</a>. 
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                    resistance as a screening method for most of our
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</p>
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                    transformations. During our research we discovered, that
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<p class="block-text medium-sized">
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                    this resistance gene was  
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<b>However, despite its broad distribution, the sequence is not fully optimized to the codon usage of <i>C. reinhardtii</i>. As we planned to use the paromomycin marker in our <i>C. reinhardtii</i> transformations we set out to use the most efficient part.</b>
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<a href="http://parts.igem.org/Part:BBa_K2703008">already contributed to the iGem
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</p>
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                    Registry</a>. We wanted to improve this part by changing the
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<p class="block-text medium-sized">
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                    codon usage and see if this improvement would end up in a
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We found an unpublished version with improved codon usage designed by <a href="https://www.chlamycollection.org/product/paphviii-pph075/">Irina Sizova</a>.
 +
In order to evaluate if this improvement would end up in
 
                     higher expression of the aminoglycoside
 
                     higher expression of the aminoglycoside
 
                     3’-phosphotransferase and therefore in a better resistance
 
                     3’-phosphotransferase and therefore in a better resistance
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                     to paramomycin. Our improved part is registered <a href="http://parts.igem.org/Part:BBa_K2984006">here</a>
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                     to paromomycin we recloned this part to meet the MoClo syntax and can be included in our <a href="https://2019.igem.org/Team:Humboldt_Berlin/Part_Collection">parts collection</a>. Our new part with improved codon usage is registered under <a href="http://parts.igem.org/Part:BBa_K2984006">BBa_K2984006</a>.
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                </p>
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                        </p>
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</div>
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                    </div>
 +
                    <div>
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                        <figure>
 +
                            <!--- IMAGE --->
 +
                            <img class="is-revealing"
 +
                                src="https://static.igem.org/mediawiki/2019/5/54/T--Humboldt_Berlin--Paro_comparision.png" alt="aphVIII codon usage comparison" />
 +
                            <figcaption>Fig. 1 - Graphical comparison of the codon usage for the old <i>aphVIII</i> (<a href="http://parts.igem.org/Part:BBa_K2703008">BBa_K2703008</a>) and our improved <i>aphVIII</i> (<a href="http://parts.igem.org/Part:BBa_K2984006">BBa_K2984006</a>) gene. Graph created with <a href="http://gcua.schoedl.de/">GCUA</a></figcaption>
 +
                        </figure>
 +
                    </div>
 +
                </div>
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                <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------>
  
 
                 <!------------------------------------------ TWO COLUMN IMG LEFT -------------------------------------------------->
 
                 <!------------------------------------------ TWO COLUMN IMG LEFT -------------------------------------------------->
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                                 src="https://static.igem.org/mediawiki/2019/3/34/T--Humboldt_Berlin--Paromomycin_structure.png"
 
                                 src="https://static.igem.org/mediawiki/2019/3/34/T--Humboldt_Berlin--Paromomycin_structure.png"
 
                                 alt="Paromomycin" />
 
                                 alt="Paromomycin" />
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                             <caption> Fig.1 - Paromomycin Molecule</caption>
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                             <figcaption> Fig.2 - Paromomycin Molecule</figcaption>
 
                         </figure>
 
                         </figure>
 
                     </div>
 
                     </div>
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                         <h3 class="headline3">Methods</h3>
 
                         <h3 class="headline3">Methods</h3>
 
                         <p class="block-text medium-sized">
 
                         <p class="block-text medium-sized">
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                             To see if the expression of the aminoglycoside 3’-phosphotransferase was increased, we performed several <a href="https://2019.igem.org/Team:Humboldt_Berlin/Experiments">electroporations to transform <i>C. reinhardtii</i></a> with the paromomycin resistance. We used the <i>C.reinhardtii</i> strain UVM 4 since it is a strain designed to express transgene constructs (Neupert et al. 2009). We compared the two paromomycin constructs with standard and improved codon usage starting with 0,5 µg DNA per electroporation sample and ascended with 0,5 µg steps up to 2 µg. For each construct and DNA mass we did three electroporations. The electroporation electrical resistance was measured for each sample. After resuspension and one day recovery in TAP medium, all samples were plated on TAP-agar plates containing a paromomycin concentration of 10 µM. After two weeks of growth, colonies corresponding to each sample were counted. Each colony of <i>C.reinhardtii</i> represents a successful transformation of the resistance and indicates the expression of the aminoglycoside 3’-phosphotransferase. By counting the amount of colonies on the plates, we could determine which construct and at which DNA mass at the time of transformation worked best.  
+
                             To see if the expression of the aminoglycoside 3’-phosphotransferase was increased, we performed several <a href="https://2019.igem.org/Team:Humboldt_Berlin/Experiments">electroporations to transform <i>C. reinhardtii</i></a> with the paromomycin resistance. We used the <i>C. reinhardtii</i> strain UVM4 since it is a strain designed to express transgene constructs (Neupert et al. 2009). We compared the old standard <i>aphVIII</i> gene with the improved codon usage starting with 0.5 µg plasmid DNA per electroporation and ascended with 0.5 µg steps up to 2.0 µg. For each construct and DNA concentration we did three electroporations. The electrical resistance was measured for each sample. After resuspension and one day recovery in TAP medium, all samples were plated on TAP-agar plates containing a paromomycin concentration of 10 µg/ml. See our <a href="https://2019.igem.org/Team:Humboldt_Berlin/Experiments">protocol section</a> for details. After two weeks of growth, paromomycin-resistant colonies were counted. Each colony of <i>C. reinhardtii</i> represents a successful transformation of the resistance gene and indicates the expression of the aminoglycoside 3’-phosphotransferase. By counting the amount of colonies on the plates, we could determine which construct and at which plasmid concentration at the time of transformation worked best.  
 
                         </p>
 
                         </p>
 
                     </div>
 
                     </div>
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                         <h3 class="headline3">Results</h3>
 
                         <h3 class="headline3">Results</h3>
 
                         <p class="block-text medium-sized">
 
                         <p class="block-text medium-sized">
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                             Counting the total number of colonies we discovered that the number of colonies was much higher for the improved plasmid version, regardless of the amount of plasmid at the time of electroporation. The colonies  of the samples using the standard usage resulted in a total amount of 175, whereas the improved plasmid version produced 665 colonies (Fig. 2). Comparing the plasmid mass we discovered that the amount of colonies does not strictly correlate to the amount of DNA used during the electroporation (Fig. 3). For the paromomycin resistance with standard codon usage we can see that the number of colonies at 1,5 µg is smaller than expected. Similarly, the amount of colonies for the improved resistance at a DNA mass of 0,5 µg is much higher than expected. The other results seem to show a tendency of increasing colony numbers with more DNA mass. Yet, further tests should be made to examine the exact effect of DNA mass during transformation for these parts. As can be seen on Fig 3.,  the mean number of colonies using the standard-plasmid is higher for 1 µg of DNA then for 1,5 µg. The same can be observed when taking the improved version into account. Here the amount of colonies for 0,5 µg ist higher than  for 1 and 1,5 µg. One explanation for the variable amount of colonies might be the inconsistency of the electroporation resistance. To see how the electroporation process affected the number of colonies their quantity was compared with the corresponding resistance. Fig. 4. depicts that the set with 1,5 µg standard plasmid was executed with a robust resistance around 570  for all 3 samples. The 1 µg set of the same plasmid shows a variable resistance but delivered more colonies. In the 2 µg standard plasmid set the resistance of the first sample dropped to 457  but the same amount of colonies as in  sample 1 of the 1,5 µg standard set were counted. With further comparison of these to sets it can be seen, that the third samples in the 1,5 µg and 2 µg sets showed similar resistance but the third sample of the second set resulted in a much higher colony number. The data behaves similar for the improved plasmid. The second sample of the first set and the third sample of the third were carried out with a resistance around 610 but for the third sample almost 34 more colonies were counted.
+
                             Counting the total number of colonies we discovered that the number of colonies was much higher for the improved <i>aphVIII</i> version. The old <i>aphVIII</i> resulted in a total of 175 colonies, whereas the improved version produced 665 colonies (Fig. 3). Comparing the plasmid concentration we discovered that the amount of colonies does not strictly correlate to the amount of DNA used during the electroporation (Fig. 4). For the paromomycin resistance with standard codon usage we can see that the number of colonies at 1.5 µg is smaller than expected. Similarly, the amount of colonies for the improved resistance at a DNA mass of 0.5 µg is much higher than expected. The other results seem to show a tendency of increasing colony numbers with more plasmid DNA. Yet, further tests should be made to examine the exact effect of plasmid concentration during transformation for these parts. As can be seen on Fig 3.,  the mean number of colonies using the standard-plasmid is higher for 1 µg of DNA then for 1.5 µg. The same can be observed when taking the improved version into account. Here the amount of colonies for 0.5 µg ist higher than  for 1.0 and 1.5 µg. One explanation for the variable amount of colonies might be the inconsistency of the electroporation resistance. To see how the electroporation process affected the number of colonies their quantity was compared with the corresponding resistance. Fig. 5. depicts that the set with 1.5 µg standard plasmid was executed with a robust resistance around 570  for all 3 samples. The 1 µg set of the same plasmid shows a variable resistance but delivered more colonies. In the 2 µg standard plasmid set the resistance of the first sample dropped to 457  but the same amount of colonies as in  sample 1 of the 1.5 µg standard set were counted. With further comparison of these to sets it can be seen, that the third samples in the 1.5 µg and 2 µg sets showed similar resistance but the third sample of the second set resulted in a much higher colony number. The data behaves similar for the improved plasmid. The second sample of the first set and the third sample of the third were carried out with a resistance around 610 but for the third sample almost 34 more colonies were counted.
 
                         </p>
 
                         </p>
 
                     </div>
 
                     </div>
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                                 src="https://static.igem.org/mediawiki/2019/a/a7/T--Humboldt_Berlin--Colony_Amount_Total.png"
 
                                 src="https://static.igem.org/mediawiki/2019/a/a7/T--Humboldt_Berlin--Colony_Amount_Total.png"
 
                                 alt="total_colonies" />
 
                                 alt="total_colonies" />
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                             <caption>Fig. 2 - Total amount of colonies counted for the standard (blue) and improved (organge) resistance</caption>
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                             <figcaption>Fig. 3 - Total amount of colonies counted for the standard (blue) and improved (organge) resistance</figcaption>
 
                         </figure>
 
                         </figure>
 
                         <figure>
 
                         <figure>
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                                 src="https://static.igem.org/mediawiki/2019/9/9d/T--Humboldt_Berlin--Colony_Amount_Mean_3.png"
 
                                 src="https://static.igem.org/mediawiki/2019/9/9d/T--Humboldt_Berlin--Colony_Amount_Mean_3.png"
 
                                 alt="mean_colonies" />
 
                                 alt="mean_colonies" />
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                             <caption>Fig. 3 - Mean colony amount for the standard (blue) and improved (orange) paromomycin resistance for different DNA mass at the time of electroporation</caption>
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                             <figcaption>Fig. 4 - Mean colony amount for the standard (blue) and improved (orange) paromomycin resistance for different DNA mass at the time of electroporation</figcaption>
 
                         </figure>
 
                         </figure>
 
                     </div>
 
                     </div>
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                 <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------>
 
                 <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------>
 
                 <!--------------------------------------- Figure and Text ------------------------------------------------>
 
                 <!--------------------------------------- Figure and Text ------------------------------------------------>
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                <div class="width-limit">
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<div class="padding-container">
 
                     <figure>
 
                     <figure>
 
                         <img class="is-revealing"
 
                         <img class="is-revealing"
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                             alt="colonies_resistance"
 
                             alt="colonies_resistance"
 
                             class="center" />
 
                             class="center" />
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                         <caption>Fig. 4 - Amount of colonies for each sample in dependency of the DNA mass and plotted with the electrical resistance of the electroporation</caption>
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                         <figcaption>Fig. 5 - Amount of colonies for each sample in dependency of the DNA mass and plotted with the electrical resistance of the electroporation</figcaption>
 
                     </figure>
 
                     </figure>
 
                 </div>
 
                 </div>
  
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              <!------------------------------------------ CENTER TEXT -------------------------------------------------->
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                <div class="width-limit">
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<div class="padding-container">
 
                     <h3 class="headline3">Discussion</h3>
 
                     <h3 class="headline3">Discussion</h3>
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                     <p class="block-text
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                     <p class="block-text medium-sized">
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                        medium-sized">
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                       <b>The experiments showed clearly, that the improved codon usage resulted in larger number of <i>C.reinhardtii</i> paromomycin-resistant colonies. This proves that the changed codon usage promotes a higher expression of the aminoglycoside 3’-phosphotransferase.</b>
−
                       The experiments showed clearly, that the improved codon usage resulted in larger number of C.reinahrdtii colonies capable of growing TAP-paromomycin-agar plates. This proves that the changed codon usage promotes a higher expression of the aminoglycoside 3’-phosphotransferase. </p>
+
</p>
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<p class="block-text
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<p class="block-text medium-sized">
−
                        medium-sized">
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Nevertheless, there are many factors that can influence the expression of the resistance gene. During  the electroporation process, transgene DNA is inserted randomly inside the genome. Depending on the regulation imposed on that genomic region, DNA transcription frequency varies, since it changes with the necessity of the genes usually coded in that same region.  This effect could be bypassed through targeted insertions, something we tried with our <a href="/Team:Humboldt_Berlin/Results">CRIPSR/Cas9 experiments.</a>
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Nevertheless, there are many factors that can influence the expression of the resistance gene. During  the electroporation process, transgene DNA is inserted randomly inside the genome. Depending on the regulation imposed on that genomic region, DNA transcription frequency varies, since it changes with the necessity of the genes usually coded in that same region.  This effect could be bypassed through targeted insertions, something we tried with our CRIPSR/Cas9 experiments(Link zur Seite).  
+
  
−
Additionally it has to be considered that for high expression, the availability of transfer RNAs is decisive. Each individual organism features a different set of tRNAs capable of matching certain types of mRNA codons. If the transgene DNA is made of codon where the organism is lacking compatible tRNA’s, the translation might be slowed down. This is what we avoided by codon optimizing the resistance gene. Since all our algae clones were exposed to the same paromomycin concentration of 10 µM the algae need a certain minimum amount of protein expression to survive the plating.
+
Additionally it has to be considered that for high expression, the availability of tRNAs is decisive. Each individual organism features a different set of tRNAs capable of matching certain types of mRNA codons. If the transgene DNA is made of codon where the organism is lacking compatible tRNA’s, the translation might be slowed down. This is what we avoided by codon optimizing the resistance gene. Since all our algae clones were exposed to the same paromomycin concentration of 10 µg/ml the algae need a certain minimum amount of protein expression to survive.
−
If the DNA is inserted inside a locus that features lower expression rates, the expression might be not high enough to withstand the pressure of selection. With the improved usage,   a locus with similar expression rate might be above that minimal amount and the algae can survive despite the locus restraints.
+
If the DNA is inserted inside a locus that features lower expression rates, the expression might be not high enough to withstand the pressure of selection. With the improved usage, a locus with similar expression rate might be above that minimal amount and the algae can survive despite the locus restraints.
 
</p>
 
</p>
 
<p class="block-text
 
<p class="block-text
 
                         medium-sized">
 
                         medium-sized">
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Besides the structure of the genome, expression of transgene DNA can be influenced by be the design of the transgene construct itself. In this case the same construct was used for bove version of the usage. The construct consist of the AR promoter, an Intron fused to beginning of the paromomycin resistance gene and the RbcS2 terminator at the end.
+
Besides the structure of the genome, expression of transgene DNA can be influenced by the design of the transgene construct itself. In this case the same construct design was used for both versions of the codon usage. The constructs consist of the AR promoter, an Intron fused to the beginning of the paromomycin resistance gene and the RbcS2 terminator at the end.
  
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In addition to genomic background and transgenic design the setup  determines the outcome of an experiment. One factor is the fitness of the cells. In preparation of our electroporation, we decided to leave out the heat shock and reduced the centrifugation speed to 12.500 rpm.
+
In addition to genomic background and transgenic design, the setup  determines the outcome of an experiment. One factor is the fitness of the cells. In preparation of our electroporation, we decided to leave out the heat shock and reduced the centrifugation speed to 1.250 rpm.
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Each pipetting step was done with high percussion to reduce damage of the cells.   
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Each pipetting step was done with high precaution to reduce damage of the cells.   
  
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A stable resistance  is  crucial for an successful transformation. If the resistance is too high the electrical pulses might not be sufficient enough to pour the membrane and transfer the DNA on the other hand, a low resistance enables stronger currents, which can lead to higher cell death rate. The resistance should vary between 400 and 600 . The majority our electroporations were performed within this range and and the effect on the number of counted colonies should be neglectable. .
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A stable resistance  is  crucial for a successful transformation. If the resistance is too high, the electrical pulses might not be strong enough to pour the membrane and transfer the DNA. On the other hand, a low resistance enables stronger currents, which can lead to higher cell death rate. The resistance should vary between 400 - 600 Ω. The majority of our electroporations were performed within this range and and its effect on the number of counted colonies should be neglectable.  
 
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This experiments clearly indicate functionality of our improved construct. For future transformations this antibiotic resistance gene can be used as a powerful screening tool boosting up the number of positive clones.  
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<b>These experiments clearly indicate enhanced functionality of our improved construct. For future transformations this antibiotic resistance gene can be used as a powerful screening tool boosting up the number of positive clones.  
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We hope that our contribution helps future iGEM Teams working with Chlamy to increase their positive results.  
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We hope that our contribution helps future iGEM Teams working with Chlamy to increase their positive results.</b>
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<h3 style="text-align:center" , class="headline3">Sources</h3>
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Sizova, I., Fuhrmann, M., & Hegemann, P. (2001). A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene, 277(1-2), 221-229.
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Neupert, J., Karcher, D., & Bock, R. (2009). Generation of Chlamydomonas strains that efficiently express nuclear transgenes. The Plant Journal, 57(6), 1140-1150.
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Latest revision as of 20:58, 21 October 2019

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Improve

Introduction

Paromomycin belongs to a group of aminoglycoside antibiotics such as neomycin or dibekacin. These aminoglycosides are capable of inhibiting the eukaryotic translation, by binding within the large and small subunit of the 80S ribosome. The bacteria Streptomyces rimosus carries the aminoglycoside 3’-phosphotransferase encoded in the so-called aphVIII gene. This enzyme inhibits paromomycin by transferring the gamma-phosphate of ATP to the hydroxyl group in 3’ position of the paromomycin molecule and allows the carrier of the gene to develop a resistance to paromomycin. The aphVIII gene is a commonly used transformation marker in green algae and established for the use in C. reinhardtii (Sizova et al. 2001) and was submitted to the registry last year as BBa_K2703008.

However, despite its broad distribution, the sequence is not fully optimized to the codon usage of C. reinhardtii. As we planned to use the paromomycin marker in our C. reinhardtii transformations we set out to use the most efficient part.

We found an unpublished version with improved codon usage designed by Irina Sizova. In order to evaluate if this improvement would end up in higher expression of the aminoglycoside 3’-phosphotransferase and therefore in a better resistance to paromomycin we recloned this part to meet the MoClo syntax and can be included in our parts collection. Our new part with improved codon usage is registered under BBa_K2984006.

aphVIII codon usage comparison
Fig. 1 - Graphical comparison of the codon usage for the old aphVIII (BBa_K2703008) and our improved aphVIII (BBa_K2984006) gene. Graph created with GCUA
Paromomycin
Fig.2 - Paromomycin Molecule

Methods

To see if the expression of the aminoglycoside 3’-phosphotransferase was increased, we performed several electroporations to transform C. reinhardtii with the paromomycin resistance. We used the C. reinhardtii strain UVM4 since it is a strain designed to express transgene constructs (Neupert et al. 2009). We compared the old standard aphVIII gene with the improved codon usage starting with 0.5 µg plasmid DNA per electroporation and ascended with 0.5 µg steps up to 2.0 µg. For each construct and DNA concentration we did three electroporations. The electrical resistance was measured for each sample. After resuspension and one day recovery in TAP medium, all samples were plated on TAP-agar plates containing a paromomycin concentration of 10 µg/ml. See our protocol section for details. After two weeks of growth, paromomycin-resistant colonies were counted. Each colony of C. reinhardtii represents a successful transformation of the resistance gene and indicates the expression of the aminoglycoside 3’-phosphotransferase. By counting the amount of colonies on the plates, we could determine which construct and at which plasmid concentration at the time of transformation worked best.

Results

Counting the total number of colonies we discovered that the number of colonies was much higher for the improved aphVIII version. The old aphVIII resulted in a total of 175 colonies, whereas the improved version produced 665 colonies (Fig. 3). Comparing the plasmid concentration we discovered that the amount of colonies does not strictly correlate to the amount of DNA used during the electroporation (Fig. 4). For the paromomycin resistance with standard codon usage we can see that the number of colonies at 1.5 µg is smaller than expected. Similarly, the amount of colonies for the improved resistance at a DNA mass of 0.5 µg is much higher than expected. The other results seem to show a tendency of increasing colony numbers with more plasmid DNA. Yet, further tests should be made to examine the exact effect of plasmid concentration during transformation for these parts. As can be seen on Fig 3., the mean number of colonies using the standard-plasmid is higher for 1 µg of DNA then for 1.5 µg. The same can be observed when taking the improved version into account. Here the amount of colonies for 0.5 µg ist higher than for 1.0 and 1.5 µg. One explanation for the variable amount of colonies might be the inconsistency of the electroporation resistance. To see how the electroporation process affected the number of colonies their quantity was compared with the corresponding resistance. Fig. 5. depicts that the set with 1.5 µg standard plasmid was executed with a robust resistance around 570 for all 3 samples. The 1 µg set of the same plasmid shows a variable resistance but delivered more colonies. In the 2 µg standard plasmid set the resistance of the first sample dropped to 457 but the same amount of colonies as in sample 1 of the 1.5 µg standard set were counted. With further comparison of these to sets it can be seen, that the third samples in the 1.5 µg and 2 µg sets showed similar resistance but the third sample of the second set resulted in a much higher colony number. The data behaves similar for the improved plasmid. The second sample of the first set and the third sample of the third were carried out with a resistance around 610 but for the third sample almost 34 more colonies were counted.

total_colonies
Fig. 3 - Total amount of colonies counted for the standard (blue) and improved (organge) resistance
mean_colonies
Fig. 4 - Mean colony amount for the standard (blue) and improved (orange) paromomycin resistance for different DNA mass at the time of electroporation
colonies_resistance
Fig. 5 - Amount of colonies for each sample in dependency of the DNA mass and plotted with the electrical resistance of the electroporation

Discussion

The experiments showed clearly, that the improved codon usage resulted in larger number of C.reinhardtii paromomycin-resistant colonies. This proves that the changed codon usage promotes a higher expression of the aminoglycoside 3’-phosphotransferase.

Nevertheless, there are many factors that can influence the expression of the resistance gene. During the electroporation process, transgene DNA is inserted randomly inside the genome. Depending on the regulation imposed on that genomic region, DNA transcription frequency varies, since it changes with the necessity of the genes usually coded in that same region. This effect could be bypassed through targeted insertions, something we tried with our CRIPSR/Cas9 experiments. Additionally it has to be considered that for high expression, the availability of tRNAs is decisive. Each individual organism features a different set of tRNAs capable of matching certain types of mRNA codons. If the transgene DNA is made of codon where the organism is lacking compatible tRNA’s, the translation might be slowed down. This is what we avoided by codon optimizing the resistance gene. Since all our algae clones were exposed to the same paromomycin concentration of 10 µg/ml the algae need a certain minimum amount of protein expression to survive. If the DNA is inserted inside a locus that features lower expression rates, the expression might be not high enough to withstand the pressure of selection. With the improved usage, a locus with similar expression rate might be above that minimal amount and the algae can survive despite the locus restraints.

Besides the structure of the genome, expression of transgene DNA can be influenced by the design of the transgene construct itself. In this case the same construct design was used for both versions of the codon usage. The constructs consist of the AR promoter, an Intron fused to the beginning of the paromomycin resistance gene and the RbcS2 terminator at the end. In addition to genomic background and transgenic design, the setup determines the outcome of an experiment. One factor is the fitness of the cells. In preparation of our electroporation, we decided to leave out the heat shock and reduced the centrifugation speed to 1.250 rpm. Each pipetting step was done with high precaution to reduce damage of the cells. A stable resistance is crucial for a successful transformation. If the resistance is too high, the electrical pulses might not be strong enough to pour the membrane and transfer the DNA. On the other hand, a low resistance enables stronger currents, which can lead to higher cell death rate. The resistance should vary between 400 - 600 Ω. The majority of our electroporations were performed within this range and and its effect on the number of counted colonies should be neglectable.

These experiments clearly indicate enhanced functionality of our improved construct. For future transformations this antibiotic resistance gene can be used as a powerful screening tool boosting up the number of positive clones. We hope that our contribution helps future iGEM Teams working with Chlamy to increase their positive results.


Sources

  1. Sizova, I., Fuhrmann, M., & Hegemann, P. (2001). A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene, 277(1-2), 221-229.
  2. Neupert, J., Karcher, D., & Bock, R. (2009). Generation of Chlamydomonas strains that efficiently express nuclear transgenes. The Plant Journal, 57(6), 1140-1150.
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