Here we share a few key results that we accomplished over the course of the summer as well as some ideas about experiments that we will pursue in the future.
As we wanted to express different genes such as mRuby2, ADP1, and GSH1 under the CCP1 promoter, we decided to use a modular cloning method to assemble our plasmids so we could swap out parts easily. We ultimately decided to use the MoClo Yeast ToolKit (YTK) is a collection of 96 standardized and characterized S. cerevisiae parts that can be used for modular and multi-part assembly of DNA constructs (Lee et. al 2015).
We were fortunate enough to receive a copy of the YTK from Zijay Tang, a research fellow at the Wyss Institute who also gave us advice on the design of our S. cerevisiaeS. cerevisiae and E. coli co-culture patch design and used the same tool kit in the construction of his Syn-SCOBY system (Gilbert et. al 2021).
Using the level 0 backbone pYTK_001 (BBa_K4320006), we were able to successfully assemble parts BBa_K4320000 through BBa_K4320005 into level 0 expression plasmids which we cloned in E. coli and sequence verified using Plasmidsaurus full plasmid sequencing services.
We then moved on to the construction of level 1 parts, BBa_K4320021 through BBa_K4320026, using the level 1 backbone pYTK_096 (BBa_K4320007), as well as pYTK_097 (BBa_K4320008) and pYTK_098 (BBa_K4320009) which were backbones we assembled using parts from the YTK for transformation into S. cerevisiae. However, ultimately we did not have enough time to completely assemble and verify these parts.
We were able to verify the pJUMP26-1A plasmid, which included superfolder GFP (sfGFP) from the iGEM distribution kit using full-plasmid sequencing from Plasmidsaurus.
We, unfortunately, did not have much luck cloning our E. coli parts as we had large gene sequences that needed to be synthesized. However, we did successfully clone and verify our level 0 acsAB, which was a 3-part + vector assembly of multiple gBlocks using the Golden Gate Assembly method. We transformed acsAB into NEB 5-alpha competent E. coli cells, screened for cells based on antibiotic resistance, and verified its sequence by full-plasmid sequencing through Plasmidsaurus.
We plan to try to assemble acsC-RBS-acsD again. We hypothesize that the low assembly efficiency is due to the fact that we are attempting a 5-part + vector assembly and have yet to optimize Golden Gate conditions. We plan to continue trying to find optimal conditions for our assembly.
We successfully cloned and verified most of our level 0 parts, including CCP1 (BBa_K4320000), ADP1 (BBa_K4320002), GSH1 (BBa_K4320003), and cyc1 (BBa_K4320005). To accomplish this, we used Golden Gate Assembly to assemble our parts and transformed them into NEB 5-alpha competentE. coliE. coli cells, screened for cells based on antibiotic resistance, and verified their sequence by full-plasmid sequencing through Plasmidsaurus. We were also able to verify many of the Dueber YTK Toolkit parts to be used for building our level 1 parts including pYTK002 (ConLS), pYTK032 (mTurquoise2), pYTK053 (tADH1), pYTK067 (ConR1), pYTK075 (LEU2), and pYTK083 (AmpR-ColE1). We did this by assembling a plasmid of many of these parts, transforming this plasmid into NEB 5-alpha competentE. coliE. coli cells, screened for ampicillin resistance, and verified its sequence by sequencing.
We successfully performed several repeats of a S. cerevisiae transformation of a test plasmid from the YTK toolkit, such as pYTK096 (BBa_K4320007) from the Dueber YTK Toolkit. This plasmid was successfully transformed into the common S. cerevisiae working strain BY4742.
However, we failed to clone other plasmids into S. cerevisiae. We tried these transformations once and plan to try again. Assuming our constructs are correct, we hypothesize that our transformation conditions are not optimal, particularly as we noted that cell density on our control plates was very high.
Lee ME, Deloache WC, Cervantes B, Dueber JE (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth Biol. 4(9):975–986. https://doi.org/10.1021/sb500366v
Gilbert C, Tang TC, Ott W, Dorr BA, Shaw WM, Sun GL, Lu TK, Ellis T (2021). Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat. Mater. 20, 691–700. https://doi.org/10.1038/s41563-020-00857-5