Endy:Notebook/Gemini

Application

Problems to solve with GFP-lacZ fusion

The goal is to highlight an operating regime in which Gemini is uniquely capable, meaning that either LacZ or GFP alone would be insufficient. It is hypothesized that the dual reporter is uniquely capable when both population and single cell measurements are desired, but gene expression is weak due to either a low copy plasmid and / or weak RBS and promoter. With this in mind, the picture to the left shows the utility of the reporter: it can report weakly expressed genes using the plate reader and LacZ where type GFP expression is undetectable. Yet, it can also be used for single cell analysis with GFP and microscopy, where wild type LacZ could not.

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Precedent

Full length GFP-lacZ fusion

A GFP-LacZ fusion dual reporter has been built and shown to encapsulate the positive properties of LacZ and GFP Hwang, 2006. This dual reporter was designed for the study of mammalian cells, specifically cell lineage within the embryo and stem cells. With this in mind, is not optimally designed for use by the synthetic biology community for three reasons.

• It is optimized for mammalian cells, rather than common chassis such as E. Coli or Yeast
• It is not bio-bricked
• It is hard to clone because it uses the full LacZ gene

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Design considerations

LacZ omega (blue), lacZalpha (red), N-terminal residue (yellow), C-terminal residue LacZ alpha (green) courtesy of Joey Davis

Evaluation of tetrameric lacZ structure shows N-termini of two adjacent lacZ alpha fragments in contact (or at the very least are in close proximity), whereas the C-termini point out into solution. Given this consideration, complimentation with C-terminal LacZa fusion to the N-terminal of GFP seems more likely to work than N-terminal LacZ fusion. That said, the first 12 residues of lacZ are not resolved in the structure. So, it may very well by that N-terminal additions are perfectly acceptable; we know from Hwang et al that LacZ monomers appear able to form a tetramer with GFP fused to the N-terminal.

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Designs

BBa E0050

BBa E0051

Two designs utilizing the alpha fragment of beta-gal were built. The first is N-terminal of LacZa to C-terminal of GFP fusion (BBaE0050, shown left). This is similar to the design of Hwang et al (2006) except for the fact that it does not have a linker. This design did not work. A linker was also added to the C-terminal of LacZa fusion to N-terminal GFP (part BBaE0051, right). The linker is glycine/serine rich for flexibility with a few charged residues to aid in solubility. This design is characterized below.

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Data

Florescence

Florescence measurements with plate reader

Comparative measurements between two inoculations

Data follow the expect trend: florescence decreases with respect to promoter strength. For promoters 115, 113, and 112 florescence is barely detectable on the plate reader relative to the controls, and there is a substantial decline is signal strength below the strongest promoter.

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LacZ

MUG assay

Xgal

MUG assay indicates beta-gal activity is identical for the three strongest promoters. Xgal plates show that 1) MUG assay correctly indicates beta-gal activity for the three strongest promoters (J23119, 101, and 106), 2) output for the moderately strong promoter J23115 is indistinguishable from from 119, 101, and 106 via Xgal plates, but is less detectable via MUG 3) J23113 and 112 are consistently undetectable. This is reasonable, as the activity of 112 and 113 are 1 and 21 au respectively, relative to 387 for 115, 1185 for 106, 1791 for 101, with all measured relative to 119, the strongest family member Promoter family.

Key points to make

Within a certain, previously defined operating regime:

1. Wild type GFP is insufficient because it cannot be detected via plate reader

• We know Gemini cannot be detected on the plate reader for weak promoters because the signal strength is too low
• We need to confirm that this is true for wild type GFP as well
  • Therefore, we are getting controls for GFP built.
  • If wild type GFP can be visualized, then why ever use Gemini?

2. LacZ alone is insufficient because it cannot be detected via microscopy

• No analysis needed to prove this point

3. Gemini produces a signal that can be detected by both mediums in domain in which either one would be insufficient

• LacZ activity is detectable via plate reader when GFP is not
  • Need to demonstrate that LacZ is detectable (with MUG assay) and GFP is not (with control)
• Over the same regulatory domain, GFP still is detectable via microscopy and LacZ is obviously not
  • Need to show that GFP is indeed detectable via microscopy.

Plan

1. Show : wild type GFP is insufficient because it cannot be detected via plate reader

• Confirm wild type GFP cannot be detected on the plate reader for weak promoters
  • Therefore, we are getting controls for GFP built.

2. Show : Gemini produces a signal that can be detected by both mediums in domain in which either one would be insufficient

• LacZ activity is detectable via plate reader when GFP is not
  • Need to demonstrate that LacZ is detectable (with MUG assay) and GFP is not (with control)
    • Need to optimize MUG assay: protocol, cell growth / OD
• Over the same regulatory domain, GFP still is detectable via microscopy and LacZ is obviously not
  • Need to show that GFP is indeed detectable via microscopy

3. Additional benchmarking : test Gemini against wild type LacZa and LacZ

• I don't think a promoter domain is necessary for this

4. Sequencing :

• Optimize prep for low copy plasmid
• Use VF2 primer

Acknowledgments

It was designed by Joey Davis and Austin Che at MIT. The promoter-RBS variants used to generate a transfer function for Gemini were built by Austin Che and Justin Buck at MIT. The controls for benchmarking performance of Gemini are being constructed by Ginko Bio-works.

Possible extension

A better design, maybe? Maybe not.