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

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                         <h3 class="headline3">1. Establishing Chlamy in the iGEM competition</h3>
 
                         <h3 class="headline3">1. Establishing Chlamy in the iGEM competition</h3>
 
                         <p class="block-text medium-sized">
 
                         <p class="block-text medium-sized">
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                           <OL> <a href="#GoldenGate"><h3 style="color:black"></h3></a>
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                           <UL> <a href="#GoldenGate"><h3 style="color:black"></h3></a>
 
                           <LI>  <a href="#GoldenGate"><h3 style="color:black">1.1 Golden Gate Modular Cloning for <i>Chlamydomonas reinhardtii</i></h3></a>
 
                           <LI>  <a href="#GoldenGate"><h3 style="color:black">1.1 Golden Gate Modular Cloning for <i>Chlamydomonas reinhardtii</i></h3></a>
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                           <LI> <a href="#GoldenGate"><h3 style="color:black">1.2 Universal Construction</h3></a>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.2. Selection cassette construction</h3></a>
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                          <OL>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.3. Experimental plan to test the functionality of our design</h3></a>
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                          <LI>  <a href="#ChlamyiGEM"><h3 style="color:black">1.2.1 Level 0-RFP Vector Design</h3></a>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.4 Protein expression analysis - testing our transgenic proteins</h3></a>
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                          <LI>  <a href="#ChlamyiGEM"><h3 style="color:black">1.2.2 Level 0 Parts Design</h3></a>
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                           <UL>
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                          <LI>  <a href="#ChlamyiGEM"><h3 style="color:black">1.2.3 Level 1 Vector Design</h3></a>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.4.1 Promoter-comparison-tests</h3></a>
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                          <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.2.4 Selection cassette construction</h3></a>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.4.2 Secretion signal</h3></a>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.2.5 Experimental plan to test the functionality of our design</h3></a>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.4.3 PtxD - phosphite decarboxylase</h3></a>
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                          </OL>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.4.4 Cas9/sgRNA-mediated site-directed mutagenesis</h3></a
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.3 Protein expression analysis - testing our transgenic proteins</h3></a>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.4.5 Modeling photoautotrophic growth of Chlamy</h3></a>
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                           <OL>
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                           </UL>                     
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.3.1 Promoter-comparison-tests</h3></a>
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                           </UL>                       
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.3.2 Secretion signal</h3></a>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.3.3 PtxD - phosphite decarboxylase</h3></a>
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.3.4 Cas9/sgRNA-mediated site-directed mutagenesis</h3></a
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                           <LI> <a href="#ChlamyiGEM"><h3 style="color:black">1.3.5 Modeling photoautotrophic growth of Chlamy</h3></a>
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                           </OL>                     
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−
                           </OL>                       
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                     </p>
 
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                         <p>
 
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                            To synthesize and assemble the desired genetic elements, we applied the Type IIS “Golden Gate” cloning strategy (Engler et al., 2008). We used the Modular Cloning (MoClo) toolkit optimized for C. reinhardtii (Crozet et al., 2018), which follows the MoClo syntax of the plant synthetic biology community (Patron et al., 2015).
+
                          To synthesize and assemble the desired genetic elements, we applied the Type IIS “Golden Gate” cloning strategy (Engler et al., 2008). We used the Modular Cloning (MoClo) toolkit optimized for <i>C. reinhardtii</i> (Crozet et al., 2018), which follows the MoClo syntax of the plant synthetic biology community (Patron et al., 2015). Type IIS restriction enzymes cut outside their recognition sites, making them useful in this cloning method for consecutive assembly of fragments. Through the restriction, overhangs are formed which allow the fusion of said genetic fragments to complementary overhangs of the syntax and thereby determine the order of each in a transcriptional unit (Figure 1). These fusion sites allow for the assembly of several fragments in the right order in just one cloning reaction. The used MoClo-kit offers ten different options for the positioning inside a L1 plasmid which are defined by the parts’ functions.  
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                        <p>
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                            Type IIS restriction enzymes (<i>Bpi</i>I; <i>Bsa</i>I) cleave outside of their recognition site leaving a four base pair overhang also called a fusion site (Engler, Kandzia, & Marillonnet, 2008). These fusion sites are determined by the used syntax. Placing restriction- and fusion sites in front of the 5’ beginning and after the 3’ end of a desired DNA fragment in an inverse orientation allows the ligation of DNA fragments with compatible fusion sites (Weber et al., 2011). Thus, the required order of sequences is defined and it is possible to assemble multiple fragments at the same time (Weber et al., 2011).
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                         </p>
 
                         </p>
 
                         <br />
 
                         <br />
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                         <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Pverview of the hierarchical and modular cloning system"/>
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                         <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system"/>
 
                         <p style="font-size:12px">
 
                         <p style="font-size:12px">
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                             <b>Fig. 1</b><figcaption> Universal MoClo fusion sites. 12 fusion sites (shown in green) generated in 2015 (Patron, 2015) for the seamless fusion of up to 10 different level 0 parts containing overlapping fusion sites. In general, the fusion sites are grouped into 5’ untranslated regions including promoter sequences (grey), the translated coding sequence CDS (blue) followed by  3’ UTRs ending with a terminator (orange). Within these types, various parts (e.g. three different coding sequences) can be designed and combined into one transcription unit when cloned together into a L1 vector. The bold ATG within the B3 fusion site sets the transcription start.</figcaption>
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                             <b>Fig. 1. Universal MoClo fusion sites. </b><figcaption> 12 fusion sites (Patron, 2015) for the seamless fusion of different level 0 parts. In general, the fusion sites are grouped into 5’ untranslated regions including promoter sequences (grey), the translated coding sequence CDS (blue) followed by  3’ UTRs ending with a terminator (orange). Within these types, various parts (e.g. three different coding sequences) can be designed a<nd combined into one transcription unit when cloned together into a L1 vector. The bold ATG within the B3 fusion site sets the transcription start.</figcaption>
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                      </div>
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                      <div>
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                        <p>
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                            Within the MoClo syntax, there are three different cloning vectors, level 0, 1 and 2 (referred to as “L0”, “L1” and “L2”, respectively). L0 vectors carry one basic genetic fragment or part, L1 vectors are assembled fragments creating a                      transcriptional unit and L2 are multigenic constructs. Construction of an L0-part is done by flanking a gene of interest with the specific fusion site and the recognition site of BpiI by a PCR reaction. Upon digestion by BpiI it can be inserted into a previously digested L0-backbone. To then clone it into a L1-backbone, it is digested by BsaI, revealing the fusion sites for its assembly in a transcription unit. Lastly, a fusion of several transcription units (L1) into a L2 multigenic device is possible with the MoClo syntax.
 
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                    <div>
 
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                        <p>
 
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                            The MoClo toolkit for Chlamydomonas consists of three different cloning vectors called level 0, 1 and 2. They are used in consecutive assembly steps. Level 0 (referred to hereafter as “L0”) destination vectors contain a selection marker gene, such as lacZ or RFP, surrounded by two restriction sites; the inner being BpiI, the outer BsaI. It is possible to insert gene parts into L0 plasmids such as promoters, coding sequences or UTRs with specific fusion sites and surrounded by BpiI restriction sites. The construct is a “level 0 part”, which is flanked by the syntax-specific fusion sites and a BsaI recognition site. The designated fusion sites (shown in green) determine the modules’ cloning position in a Level 1 (“L1”) plasmid. The used MoClo-kit offers ten different options for the positioning inside a L1 plasmid which are defined by the parts’ function.
 
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<p>
 
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                        When using these fusion sites, it is crucial to maintain the reading frame of the coding sequence, since a four base insertion can change the triplet code. Since the fusion site B3 contains the start codon ATG, the ATG within the original coding sequence needs to be inserted into this fusion site or has to be deleted. In any case, since the overhang contains four bases, it is crucial that after every element, which is part of the transcribed unit, two additional bases are integrated after the gene sequence to avoid a frame-shift, a shift in the reading frame of the following parts. To keep the native function of the protein, the selection of additional bases is not completely arbitrary and non-disturbing amino acids like alanine should be selected when possible. The stop codon TAA is not a part of any fusion site and must be attached to the end of the last part of the transcriptional unit (e.g. B5) or in front of the terminator (e.g. B5/C1, B6/C1) when creating a primer. </p>
 
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<p>
 
−
This makes it possible to correctly assemble those L0 parts onto the next level 1 plasmid (referred to hereafter as “L1”) in one step generating a transcriptional unit. Each assembly of L1 or L2 is performed in a single reaction mixed with the desired insert, the destination vector, DNA ligase and the needed type IIS restriction enzyme.  </p>
 
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                        As part of our contribution to a toolkit usable by future iGEM teams we design and construct not only the parts we intend to use on our goal of PET-degradation but several more, a L0-backbone and L1-backbone. To see more details on the design and assembly mechanism of these see the MoClo Design page.
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                             <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d9/T--Humboldt_Berlin--designfig2.png" alt="ideonella grafic" />
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                             <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d9/T--Humboldt_Berlin--designfig2.png" alt="cloning strategy" />
 
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                     <p><b>Fig. 2. Overview of the Golden Gate cloning strategy.</b><p><figcaption> Multiple basic genetic elements on a level 0 vector (Phytobricks) can be assembled to a full transcription unit on a level 1 vector. Specific fusion sites (shown in grey) and BpiI recognition sites are added to new genetic elements via PCR. To build a L0 construct the Type IIS restriction enzyme BpiI digests the PCR fragment and the L0 vector. The L0 vector, in turn, contains the recognition site for BsaI. Digestion with BsaI and ligation of several L0 parts with a L1 backbone leads to a L1 transcriptional unit. The different L1 modules of choice can then be assembled via BpiI into the final L2 construct in which no recognition sites for type IIS restriction enzymes are left.</figcaption>
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<p><b>Fig. 2</b><p><figcaption> Overview of the Golden Gate cloning strategy. Multiple basic genetic elements on a level 0 vector (Phytobricks) can be assembled to a full transcription unit on a level 1 vector. Specific fusion sites (shown in grey) and BpiI recognition sites are added to new genetic elements via PCR. To build a L0 construct the Type IIS restriction enzyme BpiI digests the PCR fragment and the L0 vector. The L0 vector, in turn, contains the recognition site for BsaI. Digestion with BsaI and ligation of several L0 parts with a L1 backbone leads to a L1 transcriptional unit. The different L1 modules of choice can then be assembled via BpiI into the final L2 construct in which no recognition sites for type IIS restriction enzymes are left.</figcaption>
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Revision as of 19:33, 30 September 2019

plasmid

Design

As previously explained on our Description page it is our aim to establish C. reinhardtii in the iGEM competition. To reach this goal we created a tool kit of various functional parts and multi-use constructs that future iGEM teams can use and optimize.

So, what is our focus?

1. Establishing C. reinhardtii as a platform in the competition

2. Working on the PET-degradation as a proof of concept

3. Building a bioreactor, in which we can cultivate C. reinhardtii and test its growth rates under different conditions

Golden Gate Modular Cloning

for Chlamydomonas reinhardtii

To synthesize and assemble the desired genetic elements, we applied the Type IIS “Golden Gate” cloning strategy (Engler et al., 2008). We used the Modular Cloning (MoClo) toolkit optimized for C. reinhardtii (Crozet et al., 2018), which follows the MoClo syntax of the plant synthetic biology community (Patron et al., 2015). Type IIS restriction enzymes cut outside their recognition sites, making them useful in this cloning method for consecutive assembly of fragments. Through the restriction, overhangs are formed which allow the fusion of said genetic fragments to complementary overhangs of the syntax and thereby determine the order of each in a transcriptional unit (Figure 1). These fusion sites allow for the assembly of several fragments in the right order in just one cloning reaction. The used MoClo-kit offers ten different options for the positioning inside a L1 plasmid which are defined by the parts’ functions.


Overview of the hierarchical and modular cloning system

Fig. 1. Universal MoClo fusion sites.

12 fusion sites (Patron, 2015) for the seamless fusion of different level 0 parts. In general, the fusion sites are grouped into 5’ untranslated regions including promoter sequences (grey), the translated coding sequence CDS (blue) followed by 3’ UTRs ending with a terminator (orange). Within these types, various parts (e.g. three different coding sequences) can be designed a

Within the MoClo syntax, there are three different cloning vectors, level 0, 1 and 2 (referred to as “L0”, “L1” and “L2”, respectively). L0 vectors carry one basic genetic fragment or part, L1 vectors are assembled fragments creating a transcriptional unit and L2 are multigenic constructs. Construction of an L0-part is done by flanking a gene of interest with the specific fusion site and the recognition site of BpiI by a PCR reaction. Upon digestion by BpiI it can be inserted into a previously digested L0-backbone. To then clone it into a L1-backbone, it is digested by BsaI, revealing the fusion sites for its assembly in a transcription unit. Lastly, a fusion of several transcription units (L1) into a L2 multigenic device is possible with the MoClo syntax.

As part of our contribution to a toolkit usable by future iGEM teams we design and construct not only the parts we intend to use on our goal of PET-degradation but several more, a L0-backbone and L1-backbone. To see more details on the design and assembly mechanism of these see the MoClo Design page.

cloning strategy

Fig. 2. Overview of the Golden Gate cloning strategy.

Multiple basic genetic elements on a level 0 vector (Phytobricks) can be assembled to a full transcription unit on a level 1 vector. Specific fusion sites (shown in grey) and BpiI recognition sites are added to new genetic elements via PCR. To build a L0 construct the Type IIS restriction enzyme BpiI digests the PCR fragment and the L0 vector. The L0 vector, in turn, contains the recognition site for BsaI. Digestion with BsaI and ligation of several L0 parts with a L1 backbone leads to a L1 transcriptional unit. The different L1 modules of choice can then be assembled via BpiI into the final L2 construct in which no recognition sites for type IIS restriction enzymes are left.