VPNWTD
Valuable Projects Nobody Wants To Do
This page is dedicated to projects and topics that would be valuable for synthetic biology efforts but are by themselves not impactful or prestigious or grant-getting enough to pursue.
Feel free to add or comment! Many brains >> fewer brains.
New orthogonal plasmids (oris, antibiotics)
pSC101, colE1 (= pBR322, pMB1, pUC variants), p15A
colE2, R6K (DIAL strains)
incW (Temme, et al.'s Nitrogen Fixation plasmids).
how many orthogonal ori's exist? Imagine part mining for orthogonal ori's. If we had a nice collection of 10 or so orthogonal, minimal ori's (possibly functional between many organisms) with different ranges of copy # (tunable by expressing replication factors at different levels), it would be tremendously enabling.
Here's a list of antibiotics used in E. coli: ampicillin (bla), kanamycin, neomycin, geneticin, chloramphenicol, zeocin, trimethoprim, spectinomycin, streptomycin, apramycin (aac) (can use 50ug/ml). This is probably not a complete list, and clearly there's a lot of overlap. Nobody has made an orthogonality grid of these yet. Also, nobody has tested what combinations of these are limiting. How many antibiotics and antibiotic resistant genes can you use in E. coli at the same time? Where are the boundaries of toxicity?
Plasmid copy # characterization
Almost nobody measures the actual plasmid copy numbers of the plasmids their systems are encoded on. We've seen huge variations in the copy numbers of supposedly "known" plasmid due to small mutations in the ori. Selection is probably driving these mutations, and blind propagation of the plasmids between people and labs is probably creating a fairly diverse phylogeny in these plasmids.
Fluorescent protein toxicity characterization
We've all been told fluorescent proteins are toxic, and we see how much slower they grow when some of them are overexpressed. Which FP's are toxic? How much? What is the mechanism of toxicity?
Also, which proteins aggregate? At what levels of expression? How is this affecting the fluorescence readout of some results?
Identify and characterize the mechanism of toxicity from various "load" issues; strong promoters, non-coding RNA
Toxicity and metabolic burden are growing challenges as synthetic systems increase in size. We need to address this problem by thorough identification and characterization of toxicity and metabolic burden.
Hypothesis: current systems are expressed at unnecessarily high levels, making toxicity a problem more than it needs to be. Not everything needs to be encoded on colE1 (high copy) and expressed with J23119.B0034 (strong expression) to be functional, but this combination will quickly lead to toxicity. One problem is that fluorescent proteins need to be expressed in large numbers to be detected by plate readers (less sensitive) and even flow cytometers (more sensitive). This lower threshold of detection is biasing us towards expressing everything at very high levels.
"KanR" cassettes in plasmids, like all cistrons, include a promoter, an RBS, and a CDS. As far as I'm aware, nobody has ever change the promoter or the RBS and correlated it with changes in the minimal inhibitory concentration (MIC) of corresponding antibiotic.
Better characterizing the antibiotic cassettes EVERYONE uses would enable us to express the minimal levels of an antibiotic resistance gene necessary for our purposes. This would enable cloning, minimize metabolic burden, and minimize the amount of antibiotic used in a cultures (bid deal considering how many antibiotic-resistance strains are around nowadays).
Minimize and properly annotate plasmids
Almost all the plasmids we have are a big mess. Most of them have huge sections of sequence that are completely unnecessary. "pSC101" origin is usually a 1kb+ chunk of DNA that includes a lot of sequence that is included only because the endonuclease sites originally used to clone it happened to exist well outside the actual ori. It would be great if someone took the time to thoroughly annotate or characterize the minimal sequence of ori's, antibiotic markers, and other prominent parts and published good sequences on them.
We need to better annotate DNA files. Even the ones that are "properly" annotated often leave out critical, higher-resolution details (promoters, RBSs, terminators, CDS's)? It's time for a new standard in annotation precision. Imagine have EVERY base-pair annotated with functionality.
When are spacers are necessary between parts
Adjacent promoters, even when they're pointed in opposite directions, will influence each other's activity. How much space (or what functional shorter part) can we place between them to insulate them from each other's activity?
What is the minimal length of a random spacer to ensure <5% transcriptional influence of 95% of sensor promoters?
Improving shitty promoters
Psal (salicylate inducible), Propionate promoter only works in BL21
Making more parts for lesser-used organisms
Well-characterized synthetic constitutive promoters for yeast, anyone?
Which sensor promoters work in which organisms? Imagine a panel of commonly-used industrial or pathogenic organisms vs. which inducible promoters (Ptac, PBAD, etc.) work in them.
Technical cytometry experiments
Test the linear range of the flow cytometer.
Test the effect of rifampicin and kanamycin on cell cultures over time.
Quantify the folding times of all the fluorescent proteins.
Make a good table for the commonly used fluorescent protein variants (sfGFP, GFPmut3b, YFP, mRFP1, mcherry, Azurite, BFP)
