Team:Virginia/Experiments
Index:
Experiments
Introduction
Due to the pandemic, we were unable to obtain lab access for the majority of the year. And when we did finally obtain lab space, access was heavily restricted; allowing only fixed groups of two or three people in the lab at a time. As such, most of our wetlab efforts were put toward the extensive planning of our assembly methods and experimental procedures as detailed below. For organization purposes, the project was split into three main sections: DNA scaffold production, BMC production, and enzyme production. The proposed methods pertaining to each of these sections are detailed in the three flow charts shown below. Clicking on the boxes in the charts will bring up more information regarding given assemblies and procedures. And scrolling and dragging while over the charts allows you to zoom in and pan around. Additionally, a key for the flow charts can be seen below in figure 1.
Fig 1. Key for the flowcharts shown below.
Scaffold Assembly
Fig 2. (Interactive) Flow chart detailing the proposed assembly methods for the DNA scaffold producing part. Scroll while hovering over the image to zoom in, click and drag to pan, and click on flow chart elements for more information regarding specific sequences and procedures.
DNA scaffolds will be produced by reverse transcription of r_oligo genes with
HIV-RTHuman Immunodeficiency Virus Reverse Transcriptase is an enzyme which allows the retrovirus to convert its genetic material from RNA to DNA in a host.
and ML-RTMurine Leukemia Reverse Transcriptase is an enzyme which allows the cancer causing retrovirus to convert its genetic material from RNA to DNA in a host.
. Two r_oligo genes will be present for every scaffold yielding two sscDNAs which are reverse complements of one another and can anneal to form a functional dsDNA scaffold. To achieve this, two primary assemblies were performed in parallel, as shown in Figure 2. The assembly depicted on the left side of the figure explains the production of the “r_oligo flipper” and its use in the production of the r_oligo genes for our templates. The assembly starts with the “GGE114” plasmid (Addgene #120731), containing an empty pSB1A3 backbone. This plasmid was used in place of the linearized pSB1A3 from the Registry due to the Registry shipping restrictions imposed by the Covid-19 epidemic. GGE114 was digested and run on an agarose gel to verify its identity. An overhang PCR reaction was performed resulting in a linearized product containing two BsmBI sites for use in Golden Gate Assembly. Due to time constraints, all other steps in this assembly have yet to be performed. However the proposed steps are detailed below.
Two gBlocks will be inserted into the “Linearized pSB1A3 Backbone with BsmBI cut sites” with a Golden Gate Assembly reaction. Due to unavoidable sequence complexity, the two gBlocks were unable to be synthesized as a single part. Together these gBlocks form a lacO regulated part inspired by the Registry’s “RFP coding device - Golden Gate Module Flipper '' (BBa_K1467400). However, unlike the Golden Gate flipper, the “r_oligo flipper” contains an HTBS sequence directly after the second BsaI site. Thus, oligonucleotides can be easily converted into r_oligo genes via a single Golden Gate Assembly. Furthermore, screening is simple as bacteria transformed with successful assemblies will not exhibit fluorescence, while unsuccessful assemblies will result in RFP production.
The “r_oligo flipper” will be used to produce eight r_oligo encoding plasmids via eight parallel Golden Gate Assemblies (“CoA 4x r_oligo”, “CoA 4x Comp r_oligo”, “Resveratrol 4x r_oligo”, “Resveratrol 4x r_oligo comp”, “CoA 6x r_oligo”, “CoA 6x comp r_oligo”, “Resveratrol 2x r_oligo”, and “Resveratrol 2x comp r_oligo”), followed by RFP based screening. Resveratrol r_oligos produce scaffolds for the 4CL and STS enzymes; CoA r_oligos produce scaffolds for the ACS and ACC enzymes. The multiplier in the names refer to the number of copies of the pathway that each scaffold will house; “comp” denotes a plasmid coding for the complementary r_oligo. Each r_oligo containing plasmid is assembled with its corresponding complement via BioBrick prefix insertion, to create composite parts that will produce self-annealing dsDNA scaffolds in the presence of reverse transcriptases. The two 4x parts, and the 6x and 2x parts will also be BioBricked together, respectively. This will result in the two final assembly products which will each produce resveratrol scaffolds and CoA scaffolds with different ratios of pathways per scaffold.
To produce these scaffolds, a part containing HIV-RT, Human Immunodeficiency Viruses Reverse Transcriptase, and ML-RT, Murine Leukemia Virus Reverse Transcriptase, will be assembled, as shown on the right side of the flow chart. Two plasmids, “pHIV_pTp66p51” (Addgene #78233) and “pMLRT” (Addgene #78234), were the basis of this assembly. Each plasmid contains SpeI and PstI restriction sites which would prevent easy use of BioBrick Assembly methods; thus, mutagenesis will be used to remove these sites from each plasmid. The pHIV mutagenesis has been started, but must be completed at a later time along with the rest of the procedures.
NEBuilder Hifi MutagenesisThe NEBuilder Hifi Mutagenesis procedure utilizes 5' exonucleases and overlapping primers to mutagenize multiple sites within a plasmid.
was utilized for the pHIV plasmid because it will allow for the removal of multiple restriction sites in a more streamlined manner. Q5 Site Directed Mutagenesis will be used for the pMLRT enzyme as it has fewer illegal restriction sites in the center of the coding sequence. After successful mutagenesis has been verified by a diagnostic gel the plasmids will be linearized with BsmBI sites via overhang PCR and assembled by a Golden Gate reaction with three additional DNA fragments. These fragments contain the promoters, lac(O), and terminator sequences for the reverse transcriptases. If a gel shows the Golden Gate assembly was successful, the cells will be inserted into “pSB1K3 containing mRFP1” (Addgene #118082) by standard assembly. The backbone swap will be confirmed by the lack of fluorescence of colonies and a gel diagnostic. This will allow the plasmid to be used in concert with the r_oligo assemblies on pSB1A3.
To assess the functionality of the assemblies, E. coli will be co-transformed with the r_oligo plasmids and the reverse transcriptase plasmids. These bacteria will be grown on medium containing ampicillin, kanamycin, and IPTG to induce scaffold formation. The scaffolds will be extracted from the cells, and run on a PAGE gel to purify and assess the ability of scaffolds to self-anneal. After extraction from the PAGE gel, the templates will be amplified in a qPCR reaction to quantify, and sequenced to verify their identity.
BMC Assembly
Fig 3. (Interactive) Flow chart detailing the proposed assembly methods for the pdu BMC producing part. Scroll while hovering over the image to zoom in, click and drag to pan, and click on flow chart elements for more information regarding specific sequences and procedures.
BMC plasmids were graciously gifted by the Warren Lab at the University of Kent in Canterbury, England. These included “pduABJKNU” and “pduABJKNUT” sequences. We verified the identities of these plasmids according to a supplemental sequence map through gel electrophoresis. Due to prior studies and modeling evidence, we conducted the rest of our studies using the pduABJKNUT plasmid
[1]J. B. Parsons, S. Frank, D. Bhella, M. Liang, M. B. Prentice, D. P. Mulvihill, and M. J. Warren, Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement, Molecular Cell, vol. 38, no. 2, pp. 305-315, 2010.
. This part included three illegal PstI restriction enzymes which we needed to remove in order to successfully utilize BioBrick prefixes and suffixes later in the procedure. NEBuilder Hifi Mutagenesis was performed on the pduABJKNUT plasmid to remove these sites. The three resultant fragments were also validated by restriction site mapping. Due to time constraints, these were the only steps completed this semester. The following steps are proposed for proper assembly of final BMC assembly products.
Further steps are required following mutagenesis to alter the pduABJKNUT part for later use. The acquired part has illegal XbaI and SpeI sites. These sites will be cut out through introduction of Golden Gate primers but will also require a new promoter, “*pduABJKNUT promoter” part and a terminator to flank the main pduABJKNUT part. The synthesized “*pduD (final product)” will be isolated in parallel to the pduABJKNUT part because the two will eventually be assembled on the same chloramphenicol backbone. From 5’ to 3’, this part includes a terminator region for the pduABJKNUT part, the pduD localization sequence along with the Zfc linker, and the terminator sequence for this pdu fusion. Our composite part will be put together through Golden Gate assembly utilizing BsmBI cut sites. This final part will include the pduABJKNUT part with the pduD fusion on a pSB1C3-Lux backbone (Addgene #109383).
We are also introducing an alternative experiment using an altered pduA protein. This customized pduA protein will have a Zfc domain to theoretically attach the DNA Scaffold in place of the pduD localization peptide. This approach will be conducted separately but through all the same methodologies of the standard procedure which utilizes the pduABJKNUT and pduD composite part.
As a final quality control step, we will be observing correct localization of our DNA scaffolds to the interior of the BMC using a green fluorescent protein. We will be isolating our synthesized “GFP final” and insert it into a synthesized pSB1T3 backbone using BsaI restriction site Golden Gate assembly. This GFP fusion has a Zif268 domain which binds a specific portion of the DNA scaffold. This pSB1T3-GFP will be co-expressed with the composite part described earlier as well as DNA Team plasmids required for DNA scaffold preparation. Upon IPTG induction, we expect our E. coli to show localized fluorescence to the interior of the BMC. We will compare our results to the results of a study that looked at images where labelled PduD-GFP localized within mCherry-labeled BMCs
[1]J. B. Parsons, S. Frank, D. Bhella, M. Liang, M. B. Prentice, D. P. Mulvihill, and M. J. Warren, Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement, Molecular Cell, vol. 38, no. 2, pp. 305-315, 2010.
. This comparison will help us to determine if we also have the steps towards successful binding of DNA scaffolds to the interior of the BMC.
Enzyme Assemblies
Fig 4. (Interactive) Flow chart detailing the proposed assembly methods for the enzyme producing parts. Scroll while hovering over the image to zoom in, click and drag to pan, and click on flow chart elements for more information regarding specific sequences and procedures.
The 4CL enzyme is responsible for the conversion of p-Coumaric acid to CoA-ester. Next, STS converts the CoA-ester into resveratrol. ACS is an enzyme which produces acetyl-CoA. Then ACC enzyme converts acetyl-CoA into malonyl-CoA which feeds into the fatty acid biosynthesis pathway, but is also needed alongside CoA-ester to create resveratrol.
Two assemblies were created in parallel and divided into 4CL/STS and ACC/ACS. To ensure the identity of the plasmids, the plasmids with 4CL, STS, and ACC were extracted, digested, then run through a gel electrophoresis. Since the Golden Gate assembly will be done with SapI, the 4CL plasmid was ordered from Addgene and processed with forward and reverse primers that contained SapI restriction sites with an overhang PCR reaction. Due to time constraints, the next steps have yet to be performed.
For the 4CL, the new PCR product with the enzyme and flanking SapI sites has to be purified, digested, then run through the gel electrophoresis. Then the pSB1A3 backbone will go through overhang PCR with forward and reverse primers containing SapI sites to form a linear backbone. Finally, the new backbone with SapI sites, the 4CL with SapI sites, and two synthesized oligos, Prefix-Promoter-LacO-RBS-ZIF268-spacer and terminator-suffix will go through a Golden Gate Assembly with SapI, forming the final 4CL assembly.
The STS enzyme also has to be processed with overhang PCR with forward and reverse primers containing BspMI restriction sites. BspMI was chosen because it was not present in the selected region of interest in the STS plasmid. Then this new PCR product with the enzyme and flanking BspMI sites has to be purified, digested, and run through gel electrophoresis. Then the pSB1A3 backbone will go through overhang PCR with forward and reverse primers containing BsaI sites to form a linear backbone. BspMI and BsaI sites will be able to bind together because they both have a four nucleotide overhang. Finally, the new backbone with BsaI sites, the STS with BspMI sites, and two synthesized oligos Prefix-Promoter-LacO-RBS-PBSII-ZF-spacer and terminator-suffix will go through a Golden Gate Assembly, forming the final STS assembly.
Using Standard Assembly, the 4CL and STS enzymes will be cut and the resulting fragments will be inserted in one of the assemblies because they are both on a pSB1A3 backbone. Then the 4CL/STS fusion on the pSB1A3 backbone will go through Standard Assembly with a pSB1T3 backbone as the final destination. To ensure the identity of this final assembly, our plasmid with 4CL/STS will be extracted, digested, then run through a gel electrophoresis.
The ACS fusion enzyme is synthesized in a pSB1A3 backbone and includes the intro (prefix-promoter-LacO-RBS-ZFB-Spacer) and outro (ACS-Terminator-Suffix) sequences. This enzyme sequence has to be purified, digested, and run through gel electrophoresis to confirm its identity.
The ACC fusion enzyme is synthesized in a pSB1A3 backbone and includes the intro (prefix-promoter-LacO-RBS-ACCalpha-terminator) and outro (promoter-LacO-RBS-ZFA-Spacer-ACCbeta-Terminator-Suffix) sequences. This enzyme sequence has to be purified, digested, and run through gel electrophoresis to confirm its identity.
Using Standard Assembly, the ACS and ACC enzymes will be cut and the resulting fragments will be inserted in one of the assemblies because they are both on a pSB1A3 backbone. Then the ACS/ACC fusion on the pSB1A3 backbone will go through Standard Assembly with a pSB1T3 backbone as the final destination. To ensure the identity of this final assembly, our plasmid with ACS/ACC will be extracted, digested, then run through a gel electrophoresis.
Final Testing
Fig 5. (Interactive) Flow chart detailing the proposed methods by which the final device will be tested. Scroll while hovering over the image to zoom in, click and drag to pan, and click on flow chart elements for more information regarding specific sequences and procedures.
The products of the three sections will be used to construct the Manifold system and analyze resveratrol yield of it as compared to plasmid scaffold and free enzyme systems. Cells with pathway enzymes bound to a plasmid scaffold via zinc-finger fusions have been shown to have increased resveratrol yields when compared to unbound enzyme systems
[2]R. J. Conrado et al., “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency,” Nucleic Acids Res., vol. 40, no. 4, pp. 1879–1889, Feb. 2012, doi: 10.1093/nar/gkr888.
. Thus, the plasmid scaffold system is an essential control in determining the capacity of Manifold to improve biosynthesis yields in comparison to the current leading systems. The resveratrol will be extracted with ethyl acetate and the yield will be quantized with HPLC. Mass spectroscopy will be used to verify the identity of the resveratrol product.
Two versions of the scaffold-BMC shell binding will be testing, a pduA zinc-finger fusion and a pduD zinc-finger fusion. A GFP zinc-finger fusion will be used in place of the pathway enzyme zinc-finger fusions. TEM will confirm the BMCs can form with the addition of a zinc-finger to a shell enzyme. Fluorescence microscopy will show that the zinc-finger fusion enzymes are able to localize to the inside of the BMCs.
Sources
[1] J. B. Parsons, S. Frank, D. Bhella, M. Liang, M. B. Prentice, D. P. Mulvihill, and M. J. Warren, Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement, Molecular Cell, vol. 38, no. 2, pp. 305-315, 2010.
[2] R. J. Conrado et al., “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency,” Nucleic Acids Res., vol. 40, no. 4, pp. 1879–1889, Feb. 2012, doi: 10.1093/nar/gkr888.
[2] R. J. Conrado et al., “DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency,” Nucleic Acids Res., vol. 40, no. 4, pp. 1879–1889, Feb. 2012, doi: 10.1093/nar/gkr888.
