Project Nemaload constructs

gistfile1.md

Project Nemaload constructs

Plan

Success entails a promoter and a protein combination.

Pan-neuronal promoters

The first set of constructs should determine the top promoters. In order to reduce the requirements for imaging conditions (i.e. freely moving), this set will use plain GFP.

1. (p)ric-19::GFP
2. (p)unc-33::GFP
3. (p)unc-76::GFP
4. (p)unc-119::GFP
5. (p)unc-51::GFP
6. (p)unc-14::GFP
7. (p)snb-1::GFP
8. (p)unc-11::GFP
9. (p)sng-1::GFP
10. (p)rpm-1::GFP
11. (p)unc-64::GFP
12. (p)jkk-1::GFP
13. (p)ehs-1::GFP
14. (p)aex-3::GFP
15. (p)rab-3::GFP

Protein combinations

Here are six candidate protein combinations:

1. RCaMP1.07 + ChR2
2. GCaMP6 + ChR2
3. GEM-GECO1 + ChR2(E123T/T159C)
4. GEM-GECO1 + ChR2(E122T/E162T)
5. GEM-GECO1 + ChR2(E123T/T159C) + NpHR
6. GEM-GECO1 + ChR2(E122T/E162T) + NpHR

Goals

Ultimately, I want every neuron in most specimens from the final strain to be modified in at least 2 ways:

• expressing a fluorescent calcium indicator
• expressing a light-activated stimulator such as channelrhodopsin (ChR2)

and possibly a third, in an additional final strain (the "uberworm"):

• expressing a light-activated inhibitor such as halorhodopsin (NpHR)

under the following constraints:

1. there is a wavelength of light that activates (causes to fluoresce) the calcium indicator without activating either the stimulator or the inhibitor. Fluorescence from the calcium indicator should vary with maximal dynamic range. (It is a important and initially non-obvious point that maximal dynamic range is anti-correlated with maximal steady-state brightness.)
2. either of:

• there is a wavelength of light that activates the inhibitor but not the stimulator, and activating the stimulator overpowers the inhibitor
• there is a wavelength of light that activates the stimulator but not the inhibitor, and activating the inhibitor overpowers the stimulator. (Energetic arguments as well as empirical data support the assertion that this condition is easier to achieve than the other one. However, it is even easier to just skip the inhibitor altogether.)

Parameter Space

Here are a list of unknowns about the final construct that need to be nailed down one way or another.

1. Choice of calcium indicator protein
2. Choice of stimulator protein
3. Choice of inhibitor protein, with "none" as an option
4. Choice of pan-neuronal promoter
5. Codon optimization
6. Construct format (single/multiple plasmids, etc.)
7. Promoter location
8. Copy number(s), i.e., DNA concentration of injection
9. Mutations

Calcium indicator

Leading candidates are RCaMP1.07, GCaMP6, and GEM-GECO1. The last construct used RCaMP1.07; it didn't work, but this could be for a variety of reasons, ranging from promoter incompatibility to an incorrect sequence, so RCaMP1.07 is still a strong possibility. GEM-GECO1 has the significant advantage of being ratiometric, but its activation wavelength is in the near-UV (380nm). While ChR2 and NpHR are advertised as sensitive primarily to blue and yellow light, the inside scoop from Ed Boyden and others is that they actually respond strongly to UV as well, thus making constraint 1 above difficult or impossible to satisfy. GCaMP6 has an absorption spectrum that overlaps significantly with ChR2, but it's claimed to be bright enough that it can be imaged with few enough photons so as not to activate ChR2.

Stimulator protein

ChR2 is the obvious choice, well used and characterized in C. elegans, but one way to attack the problem of making GEM-GECO1 work is to find a stimulator opsin that does not respond to UV. ChR2(E123T/T159C) and C1V1(E122T/E162T) are good candidates.

Inhibitor protein

NpHR is the obvious choice, well used and characterized in C. elegans, even together with ChR2. eNpHR3.0 is known to be better in mammalian neurons, but I can find no reference to its use in C. elegans. It might be worth trying.

Pan-neuronal promoter

I have a list of 16 pan-neuronal promoters from Ruvinsky et al. 2007, reproduced and reordered below. Last year I evaluated unc-119 against rab-3 and concluded the former is better. The construct currently being made at MIT uses ric-19. With unlimited molecular biology resources, it would be worth trying every one (even in the case of rab-3, controls for such variables as copy number were totally absent). However, based on my current state of knowledge, I would prioritize them in this order:

1. (p)ric-19
2. (p)unc-33
3. (p)unc-76
4. (p)unc-119
5. (p)unc-51
6. (p)unc-14
7. (p)snb-1
8. (p)unc-11
9. (p)sng-1
10. (p)rpm-1
11. (p)unc-64
12. (p)jkk-1
13. (p)ehs-1
14. (p)aex-3
15. (p)rab-3
16. (p)unc-10

Codon optimization

If de novo DNA synthesis is involved in making any of the transgenes, I assume it's worth codon-optimizing for the nematode. Otherwise, I assume it's impossible. Basically, this argues in favor of de novo DNA synthesis if nematode-optimized plasmids aren't available.

Construct format

As I understand it, there are basically three choices:

• Multiple separate plasmids, one for each transgene
• One single plasmid
  • Copy promoter region for each transgene
  • Join transgenes with stop codons, all under the control of a single promoter region

The only significant argument in favor of any of these options is that the first (separate plasmids) makes for more flexibility in tweaking copy number (see below). The construct currently being made is of this form. On the other hand, I assume that this form is the most difficult to chromosomally integrate.

Promoter location

In addition, there is some question about where to position the promoter relative to the transgene, in terms of a number of base pairs. I'm not sure if it's worth trying a lot of different options or not. I'm hoping for some expert insight here as well.

Copy number

Once all the other questions are satisfied, given a few leading plasmids (or plasmid sets), it becomes easy to experiment with the concentration of DNA injected into the worms (which is directly correlated to the copy number of transgenes in the resulting strains). The key observable here is the fluorescence of the calcium under baseline conditions as well as under a wide variety of wide-field illumination conditions (with the primary variable being wavelength and the secondary variable being illumination duration). Then I'll select the strain with the "best" profile for chromosomal integration. (How that selection is made is a bit complicated, but "maximal dynamic range" captures it to first order.)

Being able to vary the copy number of each transgene independently seems like a valuable knob, arguing in favor of separate plasmids.

Mutations

It's always possible to mutagenize any component (promoter, transgene) and see if some mutants work better. But it does lead to a sort of combinatorial explosion.