|
|
| Line 111: |
Line 111: |
| | <h3 class="headline3">Introduction</h3> | | <h3 class="headline3">Introduction</h3> |
| | <p class="block-text medium-sized"> | | <p class="block-text medium-sized"> |
| − | 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. This property allows paromomycin to be used as selection marker for C.reinhardtii. For the selection process to work, one must consider a way to implement a paromomycin resistance in Chlamydomonas. The bacteria Stretpomyces rimous carries the aminoglycoside 3’-phosphotransferase encoded in the so called aphVIII gene. This enzyme catalyses the transfer of 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 (Sizova et al. 2001). We used this resistance as a screening method for most of our transformations. During our research we discovered, that this resistance gene was already contributed to the iGem Registry. We wanted to improve this part by changing the codon usage and see if this improvement would end up in a higher expression of the aminoglycoside 3’-phosphotransferase and therefore in a better resistance to paramomycin. | + | 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. This property allows paromomycin to be used as selection marker for <i>C. reinhardtii</i>. For the selection process to work, one must consider a way to implement a paromomycin resistance in <i>C. reinhardtii</i>. The bacteria Stretpomyces rimous carries the aminoglycoside 3’-phosphotransferase encoded in the so called aphVIII gene. This enzyme catalyses the transfer of 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 (Sizova et al. 2001). We used this resistance as a screening method for most of our transformations. During our research we discovered, that this resistance gene was already contributed to the iGem Registry. We wanted to improve this part by changing the codon usage and see if this improvement would end up in a higher expression of the aminoglycoside 3’-phosphotransferase and therefore in a better resistance to paramomycin. |
| | </p> | | </p> |
| | </div> | | </div> |
| Line 128: |
Line 128: |
| | <!----------------------------TEXT----------------------------> | | <!----------------------------TEXT----------------------------> |
| | <!-----------------------------------------------------------------------> | | <!-----------------------------------------------------------------------> |
| − | <h3 class="headline3">Text Headline</h3> | + | <h3 class="headline3">Methods</h3> |
| | <p class="block-text medium-sized"> | | <p class="block-text medium-sized"> |
| − | Modified Codon Usage of Paromomycin Plasmid | + | To see if the expression was increased, we performed several electroporations to transform <i>C. reinhardtii</i> with the paromomycin resistance (Link to protocol). 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 old and new 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. |
| | </p> | | </p> |
| | </div> | | </div> |
| Line 137: |
Line 137: |
| | | | |
| | <!--------------------------------------- TWO COLUMN IMG LEFT END -----------------------------------------------> | | <!--------------------------------------- TWO COLUMN IMG LEFT END -----------------------------------------------> |
| − |
| |
| − | <!---------------------------------------------- QUOTE -------------------------------------------------------->
| |
| − |
| |
| − | <blockquote class="quote image-left">
| |
| − | <div class="quote-image-container">
| |
| − | <!-- QUOTE IMAGE -->
| |
| − | <img src="https://static.igem.org/mediawiki/2019/2/2d/T--Humboldt_Berlin--flasche_igem.png" alt="plastic bottle illustration" />
| |
| − | </div>
| |
| − | <div class="block-text big-underline big-text">
| |
| − | <!----------------------------------------------------------------------->
| |
| − | <!---------------------------- Quote Text ---------------------------->
| |
| − | <!----------------------------------------------------------------------->
| |
| − | <p><b>
| |
| − | Have the Quote Text Here - it is underlined
| |
| − | </b></p>
| |
| − | </div>
| |
| − | </blockquote>
| |
| − |
| |
| − | <!---------------------------------------------- QUOTE END ------------------------------------------------------->
| |
| | | | |
| | | | |
