<h3style="text-align: center">Why do we care?</h3>
<p>Infinite growth in finite space is impossible. Similarly, unbridled production in a closed system inevitably generates pollution due to waste. A glance at a satellite image of the Pacific Ocean offers a grim portrait of the consequences of plastic pollution on earth. The collection of plastic garbage1 washed out to sea from coastlines now occupies a surface area roughly 2 million km2. Plastics, nonetheless, have solved problems and enabled the advancement of myriad industries since their inception in 1907. Given the many essential roles filled by plastics in daily life, it is difficult to imagine a fast transition back to a plastic free world. How could synthetic biology address the seemingly paradoxical scenario we find ourselves in as of 2022?</p>
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<imgsrc="https://www.geographyrealm.com/wp-content/uploads/2018/03/map-great-ocean-garbage-patch-1.png"alt="Figure 1a from Lebreton et al. Evidence that the Great Pacific Garbage Patch is rapidly accumulating plastic. Scientific Reports. 2018, 8, 4666.">
<p><em>Figure 1a from Lebreton et al. Evidence that the Great Pacific Garbage Patch is rapidly accumulating plastic. </em>Scientific Reports<em>. 2018, <b>8</b>, 4666 </em></p>
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<p>In view of the ubiquity of plastic waste worldwide, the Uppsala PETerminator project targeted the communities most upset by plastic pollution.</p>
<h3>References</h3>
<p>1) Lebreton et al. Evidence that the Great Pacific Garbage Patch is rapidly accumulating plastic. <em>Scientific Reports</em>. 2018, <b>8</b>, 4666
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<!--<h3>Problem</h3>
<p>Initially the CDS of PETase, MHETase and LCC were ordered from Integrated DNA Technologies (IDT) and designed to be compatible with any BioBrick vector.
After the order was shipped, it was noticed that the necessary flanking regions next to the restriction sites to allow effective restriction were missing.
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<h3>Fix</h3>
<p>Fortunately, this mistake was noticed early enough to design primers which could introduce extra base pairs in both ends of the construct by annealing to the prefix (fwd) or suffix (rev). Since all the constructs initially ordered ended with the prefix and suffix one universal primer pair was able to fix the constructs via PCR. PCR primers used were:
<br><br>Forward 5’-NNNNNGGAATTCGCGGCCGCTTCTAG,
<br><br>Reverse 5’-NNNNNNCTGCAGCGGCCGCTACTA-3’.
<br><br>Further orders from IDT were made containing the necessary flaking regions.
src="https://static.igem.wiki/teams/4378/wiki/img/terminator-glasses.png">Cloning and Transformation</button>
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<h3>Design</h3>
<p>To allow proper overexpression of the different enzymes the now fixed sequences should be ligated into a pET24a vector which was modified in 2009 to be compatible with BioBrick cloning.
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<h3>Problem</h3>
<p>The transformation plates usually showed no or very few colonies after one night of incubation.
The few colonies which grew also showed in restriction analysis religation of the pET24 vector instead of the desired assembled construct.
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<h3>Fix</h3>
<p>To overcome this problem NEB® 5-alpha Competent E. coli (High Efficiency) were used.
Those showed after transformation high amounts of colonies. Screening of those colonies showed that usually 25-50% of those contained the correct assembled construct.
This showed that the cloning protocols used showed low efficiency which could be caused by enzymes with lowered activity due to suboptimal storage and handling.
Another factor which could have had influence on our results was the before mentioned PCR to fix the construct as the PCR samples needed gel purification due to undesired side products.
These purification usually resulted in low yield and impure DNA samples. The commercial cells which had a transformation efficiency 10^4 higher than our prepared cells most likely just overcame the low efficiency of the cloning procedure by its efficiency.
<p>As multiple Lab groups struggled with the use of pET24a as a vector they changed to a pSB1C3 vector containing a cassette for constitutive protein expression of the chromoprotein mRFP1 as it allowed easier screening for successful transformation by color of the colony instead of restriction analysis.
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<h3>Problem</h3>
<p>After a Lab group succeeded cloning the fixed construct into pSB1C3 they attempted protein overexpression.
Multiple tries didn’t show any sign of expression of the desired protein in SDS-PAGE analysis even though an overexpression of the T7-Polymerase in the BL21 cells was observed.
The reason for this wasn’t ultimately found.
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<h3>Fix</h3>
<p>The same expression cassette was later cloned into a pET24a vector.
With this vector which is specifically designed for overexpression a band at the desired size was observed in SDS-PAGE which shows the importance of expression vectors over vectors designed for different purposes.
