Difference between revisions of "Team:Virginia/Experiments"

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.

DNA scaffolds will be produced by reverse transcription of r_oligo genes with and . 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. 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.