Team:Virginia/Experiments
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Experiments
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Experiments
Introduction
Our team has worked exceedingly hard since the start of 2021 crafting the design, assembly methods, and experimental procedures highlighted below. The project was divided into three different sections: DNA scaffold assembly, BMC assembly, and enzyme assemblies. The flowcharts under each section detail the experimental methods used to create them. By clicking on the colored boxes in the flowcharts, extra information pertaining to the assemblies and procedures are provided. There is also a key that provides additional information.
Scaffold Assembly
DNA scaffolds were produced by reverse transcription (RT) of r_oligo genes with
HIV-RT and ML-RT. The fact that the two r_oligo genes yield two ssDNAs sequences, which are reverse complements of each other, and can anneal, allows for the production of functional dsDNA scaffolds. To attain dsDNA scaffolds, two assemblies were performed together, as highlighted in this flowchart. The first chart is detailed to show the assembly of the "r_oligo flipper". This part is extremely useful, as it aids in the production of the r_oligo genes. To make the r_oligo flipper, Addgene plasmid GGE114 with an empty pPSB1A3 backbone was acquired, digested, and run on a standard agarose gel. Upon its verification, sections of the plasmid were amplified by a PCR reaction, and the resulting linearized pSB1A3 backbone with BsmBI cut sites was used in a Golden Gate Assembly procedure.
For the Golden Gate Assembly, two gBlocks were inserted into the PCR product. The r_oligo flipper remains unique because it has an HTBS sequence directly following the second BsaI site. As an effect, oligonucleotides can be easily and efficiently converted into r_oligo genes through a Golden Gate Assembly procedure. In addition, due to the RFP insert, bacteria transformed with a successful assembly will not produce RFP, whereas bacteria not transformed successfully will produce RFP, and the colonies will be red. The addition of RFP allows for an easier and more efficient screening and detection process.
For the Golden Gate Assembly, two gBlocks were inserted into the PCR product. The r_oligo flipper remains unique because it has an HTBS sequence directly following the second BsaI site. As an effect, oligonucleotides can be easily and efficiently converted into r_oligo genes through a Golden Gate Assembly procedure. In addition, due to the RFP insert, bacteria transformed with a successful assembly will not produce RFP, whereas bacteria not transformed successfully will produce RFP, and the colonies will be red. The addition of RFP allows for an easier and more efficient screening and detection process.
After the successful production of the r_oligo flipper, it was utilized in addition with eight other DNA blocks in another Golden Gate Assembly, leading to the production of eight r_oligo genes that, when reverse transcribed, code for the DNA scaffolds CoA 4x r_oligo, CoA 4x comp r_oligo, ResV 4x r_oligo, ResV 4x comp r_oligo, CoA 6x r_oligo, CoA 6x Comp r_oligo, ResV 2x r_oligo, ResV 2x Comp r_oligo. Some quick terminology: the “2”, “4”, or “6” in the r_oligo genes signifies the number of copies of the pathway each scaffold will house, and “comp” refers to a plasmid coding for the complementary r_oligo. The different r_oligos scaffolds, ResV r_oligos and CoA r_oligos, were important for the proper binding and coordination of the enzymes as described in the 'Enzymes Assemblies' section.
Towards the end of this flowchart, BioBrick assembly ensured that each r_oligo was assembled correctly with its complement in order to create self-annealing parts that produced the dsDNA scaffolds. CoA 4x and Resv 4x were biobrick assembled together, as were CoA 6x and ResV 2x. The Biobrick assemblies resulted in the last two products, which assembled different ratios of ResV and CoA scaffolds.
Towards the end of this flowchart, BioBrick assembly ensured that each r_oligo was assembled correctly with its complement in order to create self-annealing parts that produced the dsDNA scaffolds. CoA 4x and Resv 4x were biobrick assembled together, as were CoA 6x and ResV 2x. The Biobrick assemblies resulted in the last two products, which assembled different ratios of ResV and CoA scaffolds.
As depicted in the flow chart above, the actual production of the dsDNA scaffolds was made. For this to happen, pHIVRT_pTp66p51, Human Immunodeficiency Viruses Reverse Transcriptase, and pMLRT, Murine Leukemia Virus Reverse Transcriptase, were assembled. Starting with a pSB1K3+ RFP empty backbone pSB1K3 + mRFP1 , a PCR procedure and purification led to its linearization with BsaI cut sites. This linearized piece of DNA was used as the backbone for a Golden Gate Assembly procedure; the HIV-RT and MLRT genes were inserted using three gBlocks, one for each subunit of HIV-RT and one for MLRT. After the Golden Gate Assembly, routine procedures were performed to acquire a diagnostic agarose gel of the plasmids, confirming RT in a pSB1K3 backbone.
For the RT in pSB1K3 to be used in conjunction with the r_oligo assemblies on pSB1A3, the two parts had to be ligated into the same plasmid. 3A assembly moved them into a pSB1C3 backbone, and after diagnostic digests, another round of BioBricking onto pSB1A3 backbones led to the combination of RT and the r_oligo genes (4,4, and 2,6).
BMC Assembly
Bacterial microcompartment plasmids were graciously gifted by the Warren Lab at the University of Kent in Canterbury, England, which included the “pduABJKNU” and “pduABJKNUT” sequences. Due to prior studies and modeling evidence, we opted to use the pduABJKNUT plasmid for our experiment. Its identity was verified by a supplemental sequence map through gel electrophoresis.
Two different parts of this plasmid were amplified through a PCR reaction, creating two linear DNA fragments with BsmBI sites on the ends. Once the two fragments were obtained, they were used for a Golden Gate Assembly with three PDU gBlocks and an Addgene plasmid pTU2-A-RFP, to create our composite parts, pduABD*JKNUT in pTU2with either CmR + TetR or just CmR, pduABD*JKNUT in pTU2 Alt
Enzyme Assemblies
The enzyme assemblies are detailed in the Nucleic Acids page; however, they’ll be going by numbers here. Each assembly contains different combinations of enzyme genes, and when compared to each other, the assemblies will demonstrate the greater efficiency of a full Manifold system than any of its individual components.
Assembly 1 codes for an HA Tag-ZFa-ACS fusion protein, and its assembly can be viewed by accessing the link. Assembly 2 codes for a HA Tag-ZFa-ACS protein and is differentiated from assembly 1 due to the fact that the ACC beta and ACS proteins are not fusions. The link provides further insight into the design of this assembly. Assembly 3 similarly codes for a HA Tag-ZFa-ACS fusion protein, therefore it contains the same assembly as the first enzyme, however it confers TetR as opposed to CmR. Assembly 4 similarly codes for a HA Tag-ZFa-ACS protein, therefore it contains the same assembly as the second enzyme, however it confers TetR as opposed to CmR. Assembly 5 codes for a 6xH-zif268-4Cl fusion followed by a 6xH-PBSII zinc finger-STS fusion with chloramphenicol resistance. The link can be utilized for a better visual of the assembly. Assembly 6 codes for the protein pduABJKNUT and has a pduD-ZFC fusion between pduB and pduJ and between TetR and CmR on a pTU2 ORI. In addition, everything except the CmR gene and the ORI are flanked by Tn5 transposase sites. Lastly, assembly 7 resembles assembly 6, however only contains CmR.
The enzymes were created by Golden Gate assembly reactions with BsmBI sites in addition with several other gBlocks. After the Golden Gate procedures, standard culture and plasmid extraction protocols were performed and the assemblies were confirmed by gel electrophoresis. The enzyme assemblies labeled 3, 4, 5, and 7 construct a Manifold device in which the pdu genes remain on their own plasmid. The results of these enzymes are still in the process, however be sure to check our presentation video for updates! On the other hand, the enzyme assemblies shown as 1, 2, 5, and 6 produces several versions of the Manifold device in which the pdu genes have been spliced into the genome of E. coli with Tn5 transposase. This ongoing process is still in the works, as the wet lab team is working diligently to achieve positive results.
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