Difference between revisions of "Team:Virginia/Design"

Revision as of 22:46, 26 October 2020

Manifold

MANIFOLD

Index:

Design

Our Mission

In our mission to create a device to increase metabolic flux, we decided to focus on the resveratrol pathway, one that is simple and researched. To increase pathway flux we propose the use of zinc-finger-domain-containing DNA scaffolds localized to the lumen of

bacterial microcompartments

Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40â€“200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.

(BMCs)

[1]Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.

, to which zinc finger fusion proteins capable of resveratrol biosynthesis are bound.

How Did We Design Manifold to Achieve All of This?

In order to achieve our goals, we planned to engineer our device to have four main parts. These main parts include the bacterial microcompartment (BMC), reverse transcriptase (RT), a scaffold DNA template, and scaffold proteins.

The first part is responsible for the production of the bacterial microcompartments. In this design, we propose the use of the Pdu microcompartment which, in nature, encloses enzymes producing 1,2-propanediol in Salmonella enterica. For use in this design only the proteins coding for the shell are required so that the 1,2-propanediol pathway can be replaced by the DNA-scaffolded resveratrol synthesis pathway. For the assembly of an empty Pdu microcompartment shell, it has been shown that only the PduA, PduB, PduJ, PduK, PduN, PduU, and PduT proteins are required forming what we call the PduABJKNUT.

The second part is responsible for the production of HIV reverse transcriptase and Murine leukemia that aids in the production of DNA scaffolds. While only HIV-RT is needed to produce the scaffolds, it has been seen that the co-expression of ML-RT leads to an increase in DNA production.

The third part is responsible for the RNA templates needed to utilize the enzymes needed for the production of the DNA scaffold as well as its reverse complement. These RNA templates are referred to as r_oligos and consist of the HIV-RT promoter known as the HIV Terminator-Binding Site (HBTS). This HBTS sequence is placed downstream of the scaffold zinc finger domain containing scaffold because HIV-RT travels through the template from the 3’ to 5’ direction. There are two r_oligo RNA sequences because each reverse transcriptase produces single stranded DNA so both sides need to be produced. Both of these r_oligo RNA sequences are controlled under two copies of the same promoters to control the concentrations of the scaffolds. Lastly, the produced scaffold sequence comprises of a domain where the PduD protein will bind and is followed by the 4Cl and STS binding sites.

The fourth part controls the production of the enzymes PduD, 4Cl, and STS that have an added zinc finger domain. All of these proteins are produced by the same part.

These four parts will make up the main device we call Manifold. This device will be able to form the BMC-Scaffold complex which will isolate the reveratrol production pathway from the rest of the cell. We divided these parts throughout our sub teams categorized the BMC team, DNA team, and Enzyme team. The composite parts of the subteams can be seen in the combined flow chart.

Fig 1. Figure taken from iGEM Tainan 2019 for demo purposes. Notice how the figure is much longer than it is wide, and two images are coupled together to achive this. Try to do that as well so it looks good.

What assembly method did we use in our design?

For our assembly, we mainly used the Golden Gate Assembly method. We have chosen this assembly for several reasons. The

Golden Gate Assembly method Golden Gate cloning technology relies on Type IIS restriction enzymes, first discovered in 1996. Type IIS restriction enzymes are unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, creating four base flanking overhangs.

utilizes type IIS restriction enzymes to cut the different parts and form a composite part

[3]Gearing, M. (n.d.). Plasmids 101: Golden Gate Cloning. Retrieved October 26, 2020, from https://blog.addgene.org/plasmids-101-golden-gate-cloning

. The type IIS enzymes are able to cleave outside of their recognition sites and create single-stranded overhangs with non-specific sequences. Since these overhangs are separate from the recognition sequence, they can be customized to direct assembly of multiple DNA fragments. This allows us to determine the direction of the parts and make sure that they are assembled in the correct order. Since the entry and destination vectors have recognition sites that are complementary and in opposite directions, this can create a final plasmid where there is no recognition site. Therefore, once the insert has been ligated, it cannot be cut again. As a result, the ligation process is very efficient and many fragments can be assembled in a single reaction. Additionally, the parts can be designed so that the there are shorter scars or no scars between the parts in the final construct.

How are we testing our design?

BMC Assay

To test for the BMCs, we will be using electron microscopy. After transformation with the plasmid, the E. coli will be sectioned and viewed to check for the presence of any BMCs. f BMCs are seen, the bacteria will then be transformed with the previously produced PduD-GFP fusion plasmid and the cells will be viewed under a fluorescence microscope at a resolution of 0.2 μm. In the absence of BMCs PduD has been shown to localize to the end of E. coli cells, however if they are properly binding to the BMCs then they will be distributed throughout the cytoplasm in a slightly more uniform manner. Additionally, if no fluorescence or continuously uniform fluorescence is seen, then the PduD-GFP fusion steps must be redone. If uniform, discrete patches of fluorescence are seen it can be concluded that the PduD proteins are properly binding to the BMCs and the functionality of the BMC producing DH10-β strain has now been verified and is part of this team's final deliverable.

Resveratrol Assay

To test that the resveratrol pathway can be implemented with our system, we will be splitting up the assay into three categories. The first assay will comprise of the the 10-beta competent E. coli with only the free enzymes 4Cl-ZF/STS-ZF and the combined 4Cl-ZF/STS-ZF and ACC-ZF/ACS-ZF assembly in order to test for the production of resveratrol as well as acetyl-coA and malonyl-coA which are also intermediates in the pathway. The second assay includes the scaffold and fusion enzymes, the third assay comprises of the entire Manifold system. Afterwards we will be putting these three groups into ethyl acetate to extract resveratrol. Then we will be using

HPLC High performance liquid chromatography is now one of the most powerful tools in analytical chemistry. It has the ability to separate, identify, and quantitate the compounds that are present in any sample that can be dissolved in a liquid.

to determine the resveratrol yield from the extraction

[4]Waters, Beginners Guide to Liquid Chromatography.[Online]. Available: https://www.waters.com/waters/en_US/HPLC---High-Performance-Liquid-Chromatography-Explained/nav.htm?cid=10048919. [Accessed: 26-Oct-2020].

. Finally, we will use

mass spectroscopyMass spectroscopy is an analytical technique that identifies biomolecules or proteins present in biological samples and is also useful for studies on protein–protein interactions.

to verify the identity of the resveratrol

[2]J. Rajawat and G. Jhingan, Chapter 1 - Mass spectroscopy, Data Processing Handbook for Complex Biological Data Sources, pp. 1-20, 2019.

. This assay will help us determine how effectively our parts and Manifold system can affect yield of the resveratrol pathway.

Sources

[1] J. Elbaz, P. Yin, and C. A. Voigt, Genetic encoding of DNA nanostructures and their self-assembly in living bacteria, Nature Communications, vol. 7, no. 1, 2016.

[2] J. Rajawat and G. Jhingan, Chapter 1 - Mass spectroscopy, Data Processing Handbook for Complex Biological Data Sources, pp. 1-20, 2019.

[3] M. Gearing, Plasmids 101: Golden Gate Cloning,addgene blog share science. [Online]. Available: https://blog.addgene.org/plasmids-101-golden-gate-cloning. [Accessed: 26-Oct-2020].

[4] Waters, Beginners Guide to Liquid Chromatography. [Online]. Available: https://www.waters.com/waters/en_US/HPLC---High-Performance-Liquid-Chromatography-Explained/nav.htm?cid=10048919. [Accessed: 26-Oct-2020].

@@ Line 125: / Line 125: @@
            <div class="section">
              <div class="paragraph">
-                The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of <div class="dict">bacterial microcompartments<span><img src="https://upload.wikimedia.org/wikipedia/commons/thumb/2/25/Carboxysome_and_bacterial_microcompartments.jpg/800px-Carboxysome_and_bacterial_microcompartments.jpg"/>Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40–200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.</span></div> (BMCs) with encapsulated dsDNA scaffolds <div class="ref">[1]<span>Elbaz, J., Yin, P., &amp; Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.</span></div> that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis. The lack of a versatile and reliable way to improve metabolic flux channeling, pathway orthogonality, and product yields is a major impediment to the expanded utilization of biosynthesis for the production of drugs and industrially valuable chemicals. Manifold, a platform technology that addresses this problem, consists of bacterial microcompartments (BMCs) with encapsulated dsDNA scaffolds that sequester and spatially organize, at fixed concentrations, biosynthetic enzymes presented as zinc-finger fusion proteins. Here we deliver the designs for an E. coli cell capable of synthesizing resveratrol using the Manifold platform. The Manifold platform will help lower costs and expand the applications of chemical biosynthesis.
+             In our mission to create a device to increase metabolic flux, we decided to focus on the resveratrol pathway, one that is simple and researched. To increase pathway flux we propose the use of zinc-finger-domain-containing DNA scaffolds localized to the lumen of <div class="dict">bacterial microcompartments<span><img src="https://upload.wikimedia.org/wikipedia/commons/thumb/2/25/Carboxysome_and_bacterial_microcompartments.jpg/800px-Carboxysome_and_bacterial_microcompartments.jpg"/>Bacterial microcompartments (BMCs) are organelle-like structures, consisting of a protein shell that encloses enzymes and other proteins. BMCs are typically about 40â€“200 nanometers in diameter and are entirely made of proteins. The shell functions like a membrane, as it is selectively permeable.</span></div> (BMCs) <div class="ref">[1]<span>Elbaz, J., Yin, P., &amp; Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7(1), 1-11.</span></div>, to which zinc finger fusion proteins capable of resveratrol biosynthesis are bound.
              </div>
            </div>
-           <div class="sectionTitle" id="Section 2">Section 2</div>
+           <div class="sectionTitle" id="Section 2">How Did We Design Manifold to Achieve All of This?</div>
            <div class="bar"></div>
            <div class="section">
              <div class="paragraph">
-               The invention consists of a protein shell comprising one or more proteins, one or more nucleic acid scaffolds of which there can be multiple copies, anabolic and/or catabolic enzymes specific to the desired biosynthesis pathway each containing a nucleic acid binding domain, recognition sequences for the utilized nucleic acid binding domains, nucleic acid spacers, and a linkage between the nucleic acid scaffolds and the protein shell. The protein shell (10) can take the form of any closed or open surface that comprises one or more repeating protein units (12). Examples of valid shells include bacterial microcompartments such as the Pdu, Eut, and carboxysome microcompartments, as well as modified,  but not necessarily closed, surfaces composed of mutated versions of these microcompartment shell proteins. The nucleic acid scaffolds (18) comprise multiple recognition sequences (22) and spacers (32) and can be made from any form of nucleic acid, including: deoxyribonucleic acid, ribonucleic acid, and synthetic nucleic acids such as xeno nucleic acids and peptide nucleic acids among others. These scaffolds are attached to the protein shell. The pathway enzymes are biological proteins whose exact sequences are dependent on the given use case of the invention, but which all contain a nucleic acid binding domain either internal to their structure, or at their N or C terminus.
+            In order to achieve our goals, we planned to engineer our device to have four main parts. These main parts include the bacterial microcompartment (BMC), reverse transcriptase (RT), a scaffold DNA template, and scaffold proteins.<br/></div>
+            <div class="paragraph">
+               The first part is responsible for the production of the bacterial microcompartments. In this design, we propose the use of the Pdu microcompartment which, in nature, encloses enzymes producing 1,2-propanediol in Salmonella enterica. For use in this design only the proteins coding for the shell are required so that the 1,2-propanediol pathway can be replaced by the DNA-scaffolded resveratrol synthesis pathway. For the assembly of an empty Pdu microcompartment shell, it has been shown that only the PduA, PduB, PduJ, PduK, PduN, PduU, and PduT proteins are required forming what we call the PduABJKNUT. <br/></div>
+              <div class="paragraph">The second part is responsible for the production of HIV reverse transcriptase and Murine leukemia that aids in the production of DNA scaffolds. While only HIV-RT is needed to produce the scaffolds, it has been seen that the co-expression of ML-RT leads to an increase in DNA production. <br/></div>
+              <div class="paragraph">The third part is responsible for the RNA templates needed to utilize the enzymes needed for the production of the DNA scaffold as well as its reverse complement. These RNA templates are   referred to as r_oligos and consist of the HIV-RT promoter known as the HIV Terminator-Binding Site (HBTS). This HBTS sequence is placed downstream of the scaffold zinc finger domain containing scaffold because HIV-RT travels through the template from the 3’ to 5’ direction. There are two r_oligo RNA sequences because each reverse transcriptase produces single stranded DNA so both sides need to be produced. Both of these r_oligo RNA sequences are controlled under two copies of the same promoters to control the concentrations of the scaffolds. Lastly, the produced scaffold sequence comprises of a domain where the PduD protein will bind and is followed by the 4Cl and STS binding sites. <br/></div>
+              <div class="paragraph"> The fourth part controls the production of the enzymes PduD, 4Cl, and STS that have an added zinc finger domain. All of these proteins are produced by the same part.<br/> </div>
+              <div class="paragraph">These four parts will make up the main device we call Manifold. This device will be able to form the BMC-Scaffold complex which will isolate the reveratrol production pathway from the rest of the cell. We divided these parts throughout our sub teams categorized the BMC team, DNA team, and Enzyme team. The composite parts of the subteams can be seen in the combined flow chart.<br/></div>
              </div>
              <div class="figureHolder">
@@ Line 140: / Line 146: @@
                </div>
              </div>
+            <div class="sectionTitle" id="Section 3">What assembly method did we use in our design?</div>
+            <div class="bar"></div>
+            <div class="section">
              <div class="paragraph">
-               Additionally, protein linkers are usually present between this nucleic acid binding domain and the enzyme structure to prevent inhibition of enzyme activity. However the exact linker(s) used,  if any, is(are) also dependent on the specific use case of the invention. These pathway enzymes are attached to the nucleic acid scaffolds via their nucleic acid binding domains. The nucleic acid recognition sequences (22) are unique or semi-unique sequences of nucleic acid monomers on the nucleic acid scaffolds to which the utilized nucleic acid binding domains have some degree of molecular complementarity. These nucleic recognition sequences comprise most of the scaffold and mark the locations to which the DNA binding domains of the pathway enzymes attach to the scaffolds. The nucleic acid spacers (32) are relatively short sequences of nucleic acid monomers that are also present on the nucleic acid scaffolds, between the recognition sequences. The linkage between the nucleic acid scaffolds (18) and protein shell (10) provides a means by which the nucleic acid scaffolds are bound to the protein shell through direct or multi-molecule complementarity. This linkage is found between the nucleic acid scaffolds and the protein shell. One example is through the addition of a nucleic acid binding domain (24) to one or more of the shell proteins forming a nucleic acid binding domain, shell protein fusion (14). Like the pathway enzymes, this nucleic acid binding-domain can be either internal to the shell protein structure or at its N or C terminus, where the exact placement depends on the shell protein being utilized. Alternatively, one or more intermediate proteins can be used to adhere the nucleic acid scaffolds to the shell, where the region of the protein interacting with the shell binds the shell via protein-protein complementarity (28) with a given shell protein, and the region of the protein interacting with the nucleic acid scaffold binds another recognition sequence on the nucleic acid scaffold through another nucleic acid binding domain (30). This forms a shell protein binding, nucleic acid domain fusion (26).<br/><br/><br/>
+               For our assembly, we mainly used the Golden Gate Assembly method. We have chosen this assembly for several reasons. The <div class="dict">Golden Gate Assembly method <span> Golden Gate cloning technology relies on Type IIS restriction enzymes, first discovered in 1996.
+              Type IIS restriction enzymes are unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, creating four base flanking overhangs.</span></div>utilizes type IIS restriction enzymes to cut the different parts and form a composite part <div class="ref">[3]<span>Gearing, M. (n.d.). Plasmids 101: Golden Gate Cloning. Retrieved October 26, 2020, from https://blog.addgene.org/plasmids-101-golden-gate-cloning</span></div>. The type IIS enzymes are able to cleave outside of their recognition sites and create single-stranded overhangs with non-specific sequences. Since these overhangs are separate from the recognition sequence, they can be customized to direct assembly of multiple DNA fragments. This allows us to determine the direction of the parts and make sure that they are assembled in the correct order. Since the entry and destination vectors have recognition sites that are complementary and in opposite directions, this can create a final plasmid where there is no recognition site. Therefore, once the  insert has been ligated, it cannot be cut again. As a result, the ligation process is very efficient and many fragments can be assembled in a single reaction. Additionally, the parts can be designed so that the there are shorter scars or no scars between the parts in the final construct.
+              <br/>
              </div>
            </div>
+        <div class="sectionTitle" id="Section 4">How are we testing our design?</div>
+            <div class="bar"></div>
+            <div class="section">
+              <b>BMC Assay</b><br/>
+            <div class="paragraph">
+            To test for the BMCs, we will be using electron microscopy. After transformation with the plasmid, the E. coli will be sectioned and viewed to check for the presence of any BMCs. f BMCs are seen, the bacteria will then be transformed with the previously produced PduD-GFP fusion plasmid and the cells will be viewed under a fluorescence microscope at a resolution of 0.2 μm. In the absence of BMCs PduD has been shown to localize to the end of E. coli cells, however if they are properly binding to the BMCs then they will be distributed throughout the cytoplasm in a slightly more uniform manner. Additionally, if no fluorescence or continuously uniform fluorescence is seen, then the PduD-GFP fusion steps must be redone. If uniform, discrete patches of fluorescence are seen it can be concluded that the PduD proteins are properly binding to the BMCs and the functionality of the BMC producing DH10-β strain has now been verified and is part of this team's final deliverable.
+            </div>
+            <b>Resveratrol Assay</b><br/>
+            <div class="paragraph">
+              To test that the resveratrol pathway can be implemented with our system, we will be splitting up the assay into three categories. The first assay will comprise of the the 10-beta competent E. coli with only the free enzymes 4Cl-ZF/STS-ZF and the combined 4Cl-ZF/STS-ZF and ACC-ZF/ACS-ZF assembly in order to test for the production of resveratrol as well as acetyl-coA and malonyl-coA which are also intermediates in the pathway. The second assay includes the scaffold and fusion enzymes, the third assay comprises of the entire Manifold system. Afterwards we will be putting these three groups into ethyl acetate to extract resveratrol. Then we will be using <div class="dict">HPLC <span> High performance liquid chromatography is now one of the most powerful tools in analytical chemistry. It has the ability to separate, identify, and quantitate the compounds that are present in any sample that can be dissolved in a liquid.</span></div>to determine the resveratrol yield from the extraction <div class="ref">[4]<span>Waters, Beginners Guide to Liquid Chromatography.[Online]. Available: https://www.waters.com/waters/en_US/HPLC---High-Performance-Liquid-Chromatography-Explained/nav.htm?cid=10048919. [Accessed: 26-Oct-2020].</span></div>. Finally, we will use <div class="dict">mass spectroscopy<span>Mass spectroscopy is an analytical technique that identifies biomolecules or proteins present in biological samples and is also useful for studies on protein–protein interactions.</span></div> to verify the identity of the resveratrol <div class="ref">[2]<span>J. Rajawat and G. Jhingan, Chapter 1 - Mass spectroscopy, Data Processing Handbook for Complex Biological Data Sources, pp. 1-20, 2019.</span></div>. This assay will help us determine how effectively our parts and Manifold system can affect yield of the resveratrol pathway.<br/>
+            </div>
+            </div>
+            <div class="sectionTitle" id="Section 5">Sources</div>
+            <div class="bar"></div>
+            <div class="section">
+            <div class="paragraph">
+              [1] J. Elbaz, P. Yin, and C. A. Voigt, Genetic encoding of DNA nanostructures and their self-assembly in living bacteria, Nature Communications, vol. 7, no. 1, 2016.
+            </div>
+            <div class="paragraph">
+              [2] J. Rajawat and G. Jhingan, Chapter 1 - Mass spectroscopy, Data Processing Handbook for Complex Biological Data Sources, pp. 1-20, 2019.
+            </div>
+            <div class="paragraph">
+              [3] M. Gearing, Plasmids 101: Golden Gate Cloning,addgene blog share science. [Online]. Available: https://blog.addgene.org/plasmids-101-golden-gate-cloning. [Accessed: 26-Oct-2020].
+            </div>
+            <div class="paragraph">
+              [4] Waters, Beginners Guide to Liquid Chromatography. [Online]. Available: https://www.waters.com/waters/en_US/HPLC---High-Performance-Liquid-Chromatography-Explained/nav.htm?cid=10048919. [Accessed: 26-Oct-2020].
+              </div>
+              </div>
+              <br/><br/>
        </div>
      </div>