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− | <h1>Trumpas title, per dvi eilutes</h1> | + | <h1>Selection system</h1> |
− | <h5>Izangos teksto stilius. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor | + | <h5>ISplit antibiotic – 2 plasmids system</h5> |
− | incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco
| + | <p>One of the essential parts of synthetic biology are plasmids. However, bacterial plasmid systems require a unique selection, usually an antibiotic resistance gene, to stably maintain the plasmids. As the number of different plasmid groups used in a single cell rise, the need for more selection markers grows. In addition to raising the issue of biosafety, the use of multiple antibiotic resistance genes destabilizes the functionality of the cells. To address this problem a protein granting the resistance to aminoglycoside family antibiotics, called amino 3'-glycosyl phosphotransferase (APH(3')), was split into two subunits by Calvin M. Schmidt et al. </p><p> |
− | laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit
| + | According to the obscure guidelines we split an unmodified neo gene sequence between 59 and 60 amino acid residues. Two subunits were termed α-neo and β-neo. Furthermore, we added additional termination codon at the end of an α-neo fragment for the translation to stop. No other start codons were included into the β-neo subunit as the gene was designed for toehold switch system. Despite the fact that β-neo subunit had no start codon, the split antibiotic system worked perfectly when coupled with a standard promoter and a ribosome binding site (BBa_K608002). Consequently, a split antibiotic resistance gene provides a selection system to stably maintain two different plasmids. |
− | esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident.</h5>
| + | </p><p><div class="img-cont"> |
− | <p>Phasellus et maximus purus, sit amet mollis neque. Maecenas finibus nec magna sit amet auctor. Ut sed sapien | + | <img src="http://placehold.it/800x450" alt="img"> |
− | quis quam auctor dictum. In sit amet eros fermentum, scelerisque lorem sit amet, aliquet est. Integer vitae
| + | <div class="img-label">Figure 1. Split neo gene principle scheme by M. Schmidt et al. |
− | eros quis velit hendrerit tempor. Aliquam odio nibh, vulputate id gravida vitae, pulvinar commodo urna.
| + | </div> |
− | Suspendisse potenti. Duis tristique molestie elementum. Donec risus mi, condimentum nec justo id, tempus
| + | </div></p> |
− | pretium sem. Donec quis sodales mauris. Maecenas sit amet felis sit amet quam commodo semper sed vel libero.
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− | Phasellus euismod faucibus sapien nec tempus. Aenean pulvinar sagittis turpis, non rutrum nibh vulputate
| + | <h2>Toehold switches – 4 plasmids system</h2> |
− | cursus. Phasellus suscipit enim at tincidunt vehicula. Nam imperdiet, magna id fringilla elementum, sem ex
| + | <p>In order to increase the capability of our selection system, we reasoned that a split antibiotic system should be put under a transcriptional or translational control. A. A. Green et al. presented wide range of de novo synthesized dynamic riboregulators, called toehold switches, which take advantage of RNA-mediated linear interaction to initiate RNA strand displacement. A toehold switch contains two parts: a ribosome binding site and a linker sequence, both of which are sequestered by a secondary RNA stem loop structure. The linker sequence has a start codon and functions as a link between RBS and protein sequence. |
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| + | </p> |
− | <h2>H2 title</h2>
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− | Integer ut sem mollis, scelerisque dolor vehicula, posuere massa. Praesent vitae metus ut sem finibus | + | |
− | ultrices. Aenean metus mi, fringilla at ex id, vehicula pulvinar diam. Etiam commodo ex nec vulputate
| + | |
− | facilisis. Nam efficitur sapien ac augue tincidunt dapibus. Vestibulum vitae sagittis lacus, sed sagittis | + | |
− | nibh. Duis fringilla diam vel gravida hendrerit. Ut eu nunc placerat, venenatis arcu vel, rhoncus justo.</p>
| + | |
− | <p>In consectetur, nibh at laoreet lobortis, ipsum odio gravida velit, a bibendum nibh urna vel lorem. Aliquam | + | |
− | luctus porttitor commodo. Nam efficitur rutrum erat id pharetra. Suspendisse interdum commodo egestas. Nulla
| + | |
− | sed posuere dolor. Vivamus malesuada mollis velit ac hendrerit. Suspendisse nec sem eu mauris tristique
| + | |
− | luctus.</p>
| + | |
| <div class="img-cont"> | | <div class="img-cont"> |
| <img src="http://placehold.it/800x450" alt="img"> | | <img src="http://placehold.it/800x450" alt="img"> |
− | <div class="img-label">Foto aprasymas it anim id est laborum. Sed ut perspiciatis unde omnis iste natus error | + | <div class="img-label">Figure 2. Principal toehold switch scheme by A. A. Green et al. |
− | sit voluptatem.
| + | |
| </div> | | </div> |
| </div> | | </div> |
| <h2>H2 title</h2> | | <h2>H2 title</h2> |
− | <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur non urna pharetra, rutrum nibh vitae, | + | <p>Although the linker sequence adds additional 10 amino acid residues to the peptide, we reasoned that it will not affect the function of split antibiotic. Toehold switches are unlocked when an RNA trigger binds to the 5’ end of the toehold and initiates RNA duplex formation, which releases the locked RBS and reveals linker start codon. We concluded, that if the toehold sequences were added in front of α- and β-neo gene fragments, the translation would require trigger RNA to initiate protein synthesis.</p><p> |
− | pellentesque erat. Curabitur fringilla ipsum id sapien imperdiet sagittis. Nulla vestibulum arcu neque, et
| + | Toeholds and their corresponding triggers design sequences were used as described by A. A. Green et al. with some modifications. First of all, it is important to note, that a “scar” which is made between biobrick prefix for protein coding sequences and suffix, contains a termination codon at the 3’ end. Therefore, it was necessary to use the other form of prefix for α- and β-neo genes, as the translation proceeds from one biobrick to another. Furthermore, seeing that the “scar” produced when joining two biobricks is 8 base pairs, we included an additional T nucleotide at the end of linker sequence to ensure the translation stays in frame to the α- and β-neo genes. We constructed a system, which includes two toehold riboregulators (termed toehold 1 and toehold 2) upstream of α- and β-neo genes in two different plasmids. The corresponding activating RNA triggers (name trigger 1 and trigger 2) were placed into additional two plasmids under constant expression. All the parts used together complete a 4-plasmid selection system - two distinct trigger RNAs are expressed by two different plasmids in order to unlock the translation of toehold controlled α- and β-neo peptides to form a complete amino 3'-glycosyl phosphotransferase. For this reason, if one plasmid is lost, the end product – α/β dimer APH(3') is not formed, therefore bacteria lose their antibiotic resistance. |
− | iaculis lorem vulputate interdum. Nunc accumsan velit ligula, ac euismod felis sagittis quis. Aenean varius
| + | </p> |
− | mollis aliquet. Nam sodales malesuada porttitor. In sed magna sed neque hendrerit ultrices.</p>
| + | <p><div class="img-cont"> |
− | <p>Phasellus et maximus purus, sit amet mollis neque. Maecenas finibus nec magna sit amet auctor. Ut sed sapien
| + | <img src="http://placehold.it/800x450" alt="img"> |
− | quis quam auctor dictum. In sit amet eros fermentum, scelerisque lorem sit amet, aliquet est. Integer vitae
| + | <div class="img-label"><IDET PAVEIKSLIUKA su 2 biobrick translation?> |
− | eros quis velit hendrerit tempor. Aliquam odio nibh, vulputate id gravida vitae, pulvinar commodo urna.
| + | </div> |
− | Suspendisse potenti. Duis tristique molestie elementum. Donec risus mi, condimentum nec justo id, tempus
| + | </div> |
− | pretium sem. Donec quis sodales mauris. Maecenas sit amet felis sit amet quam commodo semper sed vel libero.
| + | |
− | Phasellus euismod faucibus sapien nec tempus. Aenean pulvinar sagittis turpis, non rutrum nibh vulputate
| + | </p> |
− | cursus. Phasellus suscipit enim at tincidunt vehicula. Nam imperdiet, magna id fringilla elementum, sem ex
| + | <h2>Phage control – 5 plasmids system</h2> |
− | finibus sapien, at mollis eros ante id massa.</p>
| + | <p>The SynOri selection system circuit could be expanded by including additional transcription factor which induced the transcription of previously described RNA triggers. The fifth plasmid would house a transcription factor for the initiation of whole system. Phage modified promoter is perfect for this task, as it is activated by cI lambda peptide and repressed by cI 434 peptide with minimal leakage. Both of the RNA triggers - 1 and 2 - were placed under control of phage modified promoter. Furthermore, downstream of the trigger gene we included cI 434 repressor under constant expression to ensure minimal leakage of the promoter. The fifth plasmid was built to constantly express cI lambda – the activator of phage promoter. In the absence of this plasmid, the gene circuit cannot function, as the trigger RNA transcription is repressed by constant cI 434 expression and toehold switches lock the translation of α/β APH(3'). When the final component of the circuit is present, the cI lambda activator enhances the transcription of both RNA triggers. The transcribed triggers then unlock the translation of α/β neo peptides which form an active protein and confer the resistance to aminoglycoside family antibiotics. |
− | <p>Praesent pulvinar enim consequat dolor efficitur viverra. Curabitur tempus auctor tellus at fermentum. | + | </p> |
− | Integer ut sem mollis, scelerisque dolor vehicula, posuere massa. Praesent vitae metus ut sem finibus | + | |
− | ultrices. Aenean metus mi, fringilla at ex id, vehicula pulvinar diam. Etiam commodo ex nec vulputate | + | |
− | facilisis. Nam efficitur sapien ac augue tincidunt dapibus. Vestibulum vitae sagittis lacus, sed sagittis | + | |
− | nibh. Duis fringilla diam vel gravida hendrerit. Ut eu nunc placerat, venenatis arcu vel, rhoncus justo.</p>
| + | |
− | <p>In consectetur, nibh at laoreet lobortis, ipsum odio gravida velit, a bibendum nibh urna vel lorem. Aliquam | + | |
− | luctus porttitor commodo. Nam efficitur rutrum erat id pharetra. Suspendisse interdum commodo egestas. Nulla
| + | |
− | sed posuere dolor. Vivamus malesuada mollis velit ac hendrerit. Suspendisse nec sem eu mauris tristique
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− | luctus.</p>
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− | <p>Pellentesque gravida ipsum quam, eu vulputate elit egestas eu. Ut eros elit, ullamcorper sit amet accumsan | + | |
− | eget, mollis pretium libero. Etiam laoreet scelerisque risus, in bibendum velit dapibus at. Sed tempor dolor
| + | |
− | semper elit aliquet, et fermentum metus tristique. Nulla vel iaculis elit. Vivamus et turpis finibus,
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− | rhoncus dui mattis, blandit sem. Maecenas in risus vestibulum, luctus felis ut, malesuada dui. Aenean vitae
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− | nunc ex. Nunc vulputate orci nec erat iaculis auctor a nec lacus. Pellentesque non ligula laoreet,
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− | sollicitudin diam sit amet, dignissim mi. Duis dictum risus nulla, eu interdum nisl laoreet ut. Integer eu
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− | leo iaculis, finibus ante eget, consequat orci. Donec semper leo et posuere vulputate. Aliquam ut nisl
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− | tristique leo.</p>
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| </div> | | </div> |
| </div> | | </div> |
Plasmid copy number control
Flexible copy number control is the core of our framework, which is based on re-engineered ColE1 origin of replication.
Base of SynORI framework - ColE1 replicon
ColE1 plasmid replicon is based on two antisense RNA molecules: RNA I and RNA II.
Transcript of RNA II forms a RNA-DNA duplex and acts as a primer for DNA polymerase and for that reason is often called replication initiator.
During the transcription of RNA II several different secondary structures can form. Part of the structures are susceptible to the binding of RNA I – a shorter antisense version of RNA II. The interaction between RNA I and RNA II start upon formation of kissing-loop pairs between their anti-complementary secondary structures. If the kissing complex persists 3’ end of RNA I starts forming a zipper-like duplex with complementary single strand RNA II region. This results in replication inhibition, because primer cannot be formed anymore, which is why RNA I is often called replication inhibitor.
The main reasons why we have chosen ColE1 as base for SynORI framework was:
- It is a light system consisting of only two regulatory RNA molecules
- It is biochemically and mathematically well characterized
- Kissing-loop complex formation kinetics allows to predict plasmid group compatibility.
Picking the control type
It immediately becomes clear that in order to control the copy number of a plasmid one could simply change RNA I promoter. But there is a reason why it was never done before!
As RNA I and RNA II are two antisense molecules, changes made to sequence will affect both of them. Location of RNA I promoter coincides with the RNA II secondary structures, which are crucial to replication primer formation.
palyginimas RNR 1 ir RNR 2 (pilnos) antriniu strukturu ir parodymas kad RNR1 promotorius yra ant svarbiu RNR 2 strukturu, taip pat pazymeti kuriu butent.
Even if one could somehow manage to change the RNA I promoter to another one without disabling replication initiation, it would still not be a convenient because picking another promoter would require a large pool of resources every time.
For that reason we have decided not to change or modify RNA I promoter inside the wild type ColE1 origin of replication, but rather to disable it completely and place a copy of it next to RNA II.
Disabling the RNA I promoter
The main problem of inactivating RNA I promoter is that precautions must be taken in order not to change critical secondary structures of RNA II.
We have first acquired a priority mutation list from literature which analyses RNA polymerase binding affinity to -10 and -35 promoter structures and its dependence on point mutations, with mutations causing the largest decrease in affinity being in the top of the list.
Then, we compiled a simulative algorithm which compared every possible combination of -10, -35 mutations and then compared them to predicted RNA II secondary structures made by CoFold, a thermodynamics-based RNA secondary structure folding algorithm that takes co-transcriptional folding into account. We have picked replicon mutants prioritizing:
- Mutants that have unchanged RNA II secondary structures.
- Mutants that are highest in mutation priority list (lowest RNA polymerase affinity).
Tailoring the copy number control
Once RNA I promoter is disabled in the ColE1 origin of replication, it can be moved to another plasmid location and used as a separate unit. Also, RNA I promoter can now be changed without damaging the replication initiation.
RNA I, and consequently, the copy number of a plasmid can now be placed under virtually any signal pattern required.
We have first showed this by placing RNA I under a series of constitutive Anderson promoters and an inducible Rhamnose promoter.
We can now flexibly control the copy number of a plasmid! What comes next?
Trumpas title, per dvi eilutes
Izangos teksto stilius. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor
incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco
laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit
esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident.
Phasellus et maximus purus, sit amet mollis neque. Maecenas finibus nec magna sit amet auctor. Ut sed sapien
quis quam auctor dictum. In sit amet eros fermentum, scelerisque lorem sit amet, aliquet est. Integer vitae
eros quis velit hendrerit tempor. Aliquam odio nibh, vulputate id gravida vitae, pulvinar commodo urna.
Suspendisse potenti. Duis tristique molestie elementum. Donec risus mi, condimentum nec justo id, tempus
pretium sem. Donec quis sodales mauris. Maecenas sit amet felis sit amet quam commodo semper sed vel libero.
Phasellus euismod faucibus sapien nec tempus. Aenean pulvinar sagittis turpis, non rutrum nibh vulputate
cursus. Phasellus suscipit enim at tincidunt vehicula. Nam imperdiet, magna id fringilla elementum, sem ex
finibus sapien, at mollis eros ante id massa.
H2 title
Praesent pulvinar enim consequat dolor efficitur viverra. Curabitur tempus auctor tellus at fermentum.
Integer ut sem mollis, scelerisque dolor vehicula, posuere massa. Praesent vitae metus ut sem finibus
ultrices. Aenean metus mi, fringilla at ex id, vehicula pulvinar diam. Etiam commodo ex nec vulputate
facilisis. Nam efficitur sapien ac augue tincidunt dapibus. Vestibulum vitae sagittis lacus, sed sagittis
nibh. Duis fringilla diam vel gravida hendrerit. Ut eu nunc placerat, venenatis arcu vel, rhoncus justo.
In consectetur, nibh at laoreet lobortis, ipsum odio gravida velit, a bibendum nibh urna vel lorem. Aliquam
luctus porttitor commodo. Nam efficitur rutrum erat id pharetra. Suspendisse interdum commodo egestas. Nulla
sed posuere dolor. Vivamus malesuada mollis velit ac hendrerit. Suspendisse nec sem eu mauris tristique
luctus.
Foto aprasymas it anim id est laborum. Sed ut perspiciatis unde omnis iste natus error
sit voluptatem.
H2 title
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Curabitur non urna pharetra, rutrum nibh vitae,
pellentesque erat. Curabitur fringilla ipsum id sapien imperdiet sagittis. Nulla vestibulum arcu neque, et
iaculis lorem vulputate interdum. Nunc accumsan velit ligula, ac euismod felis sagittis quis. Aenean varius
mollis aliquet. Nam sodales malesuada porttitor. In sed magna sed neque hendrerit ultrices.
Phasellus et maximus purus, sit amet mollis neque. Maecenas finibus nec magna sit amet auctor. Ut sed sapien
quis quam auctor dictum. In sit amet eros fermentum, scelerisque lorem sit amet, aliquet est. Integer vitae
eros quis velit hendrerit tempor. Aliquam odio nibh, vulputate id gravida vitae, pulvinar commodo urna.
Suspendisse potenti. Duis tristique molestie elementum. Donec risus mi, condimentum nec justo id, tempus
pretium sem. Donec quis sodales mauris. Maecenas sit amet felis sit amet quam commodo semper sed vel libero.
Phasellus euismod faucibus sapien nec tempus. Aenean pulvinar sagittis turpis, non rutrum nibh vulputate
cursus. Phasellus suscipit enim at tincidunt vehicula. Nam imperdiet, magna id fringilla elementum, sem ex
finibus sapien, at mollis eros ante id massa.
Praesent pulvinar enim consequat dolor efficitur viverra. Curabitur tempus auctor tellus at fermentum.
Integer ut sem mollis, scelerisque dolor vehicula, posuere massa. Praesent vitae metus ut sem finibus
ultrices. Aenean metus mi, fringilla at ex id, vehicula pulvinar diam. Etiam commodo ex nec vulputate
facilisis. Nam efficitur sapien ac augue tincidunt dapibus. Vestibulum vitae sagittis lacus, sed sagittis
nibh. Duis fringilla diam vel gravida hendrerit. Ut eu nunc placerat, venenatis arcu vel, rhoncus justo.
In consectetur, nibh at laoreet lobortis, ipsum odio gravida velit, a bibendum nibh urna vel lorem. Aliquam
luctus porttitor commodo. Nam efficitur rutrum erat id pharetra. Suspendisse interdum commodo egestas. Nulla
sed posuere dolor. Vivamus malesuada mollis velit ac hendrerit. Suspendisse nec sem eu mauris tristique
luctus.
Pellentesque gravida ipsum quam, eu vulputate elit egestas eu. Ut eros elit, ullamcorper sit amet accumsan
eget, mollis pretium libero. Etiam laoreet scelerisque risus, in bibendum velit dapibus at. Sed tempor dolor
semper elit aliquet, et fermentum metus tristique. Nulla vel iaculis elit. Vivamus et turpis finibus,
rhoncus dui mattis, blandit sem. Maecenas in risus vestibulum, luctus felis ut, malesuada dui. Aenean vitae
nunc ex. Nunc vulputate orci nec erat iaculis auctor a nec lacus. Pellentesque non ligula laoreet,
sollicitudin diam sit amet, dignissim mi. Duis dictum risus nulla, eu interdum nisl laoreet ut. Integer eu
leo iaculis, finibus ante eget, consequat orci. Donec semper leo et posuere vulputate. Aliquam ut nisl
porta, sagittis tortor accumsan, pellentesque odio. Ut dolor sapien, porttitor vel ex vitae, condimentum
tristique leo.