(A) Unnatural protein test expression (5-50 mL): user provides the verified plasmids and the UP facility will provide controls.

  1. We would discuss the plasmid constructs to insure they are compatible with the additional translational components needed for your experiment.
  2. Your lab would send the UP Facility the wild-type construct and TAG disrupted construct of your choosing.
  3. The UP Facility transforms your plasmids into the correct cells with the correct machinery components including the correct controls genes for your experiment. Single colonies are selected and small-scale cultures are grown in non-inducing media. 
  4. The Facility expresses your protein (wt and ncAA-protein) on small scale (5-50 mL) in our auto-induction media optimized for protein yield and stability. Samples time points are sampled for cell growth and protein expression.
  5. Crude cell lysate is assessed by SDS-PAGE along with control expressions that incorporated your ncAA of interest into our control proteins. 
  6. The UP Facility sends you a summary write up including SDS-PAGE analysis. This should be sufficient to prove your desired ncAA-protein is being produced. See example …
  7. The residual cells from small scale expression can be shipped to if desired.

Cost: $550 set up fee. $65/amino acid.

 

(B) Unnatural protein expression optimization: This follows a test expression with controls. Limited to media, temperature and expression time variables.

  1. Following a standard test expression of your protein by the Facility you might want additional conditions assessed to improve ncAA-protein yield prior to scale up expression.
  2. In parallel with standard expression conditions the facility will express one or more variables depending on your input about your particular protein needs. Common variables for that we have altered in the past that alters ncAA-protein yield and solubility are temperature, expression time, cell type, induction method, and media composition.
  3. The Facility expresses your protein (wt and ncAA-protein) on small scale (5-50 mL) in the new conditions. Samples time points are sampled for cell growth and protein expression.
  4. Crude cell lysate is assessed by SDS-PAGE along with control expressions that incorporated your ncAA of interest into our control proteins. 
  5. The UP Facility sends you a summary write up including SDS-PAGE analysis. See example …
  6. The residual cells from small-scale expression can be shipped to if desired.

Cost: $450/optimization parameter.

 

(C) Unnatural protein expression (0.250 L – 5 L): user provides the verified plasmids and the UP facility will provide controls. This follows a test expression with controls.

  1. Following a standard test expression of your protein by the Facility you might want a larger scale expression in order to purify significant amounts of ncAA-protein for analysis and experiments. The Facility would consult with you regarding what scale is best to provide the amount of ncAA-protein needed.
  2. The UP Facility transforms your plasmids into the correct cells with the correct machinery components including the correct controls genes for your experiment. Single colonies are selected and small-scale cultures are grown in non-inducing media. 
  3. The Facility expresses your protein (wt and ncAA-protein) on large scale (0.25- 5 L). Samples time points are sampled for cell growth and protein expression.
  4. Crude cell lysate is assessed by SDS-PAGE along with control expressions that incorporated your ncAA of interest into our control proteins. 
  5. The UP Facility sends you a summary write up including SDS-PAGE analysis. See example …
  6. The cells are pelleted and shipped on dry ice when you are ready for them.

Cost: $450 set up fee. $65/L of media. 

 

(D) Unnatural amino acid incorporation screening: user provides the amino acid to be screen and the UP facility will provide controls. Screening will be done in GFP control protein.

  1. Amino acid that you want incorporated into protein is shipped to the UP Facility. 
  2. In consult with you the Facility selects RS families that have been previously assess for efficiency and permissivity with parent ncAAs.
  3. Using a fluorescent reporter assay your amino acid is screened for its ability to be incorporated with the library of functioning ncAA-RSs with negative and positive controls.
  4. The results are graft and data sent to you describing which RS members are incorporating your amino acid of interest. 

Cost: $350 set up fee. $65/amino acid.

 

(E) Unnatural amino acid synthetase library selection: user provides the amino acid to be screen and the UP facility will provide the synthetase library and selection constructs, and controls. Screening will be done in GFP control protein.

Cost: $2700 per round of selection and evaluation. Supplies billed separately

 

(F) UP facility: new user fee
The fee is for training time to set up a new user (6-7 hrs) on how to use the facility. Manditory.
Cost: $350 each

 

(G) UP facility: Unassisted Use Fee
Cost: $150/month

 

(H) UP facility: Technical assistance 
0.5 hr minimum charge
Cost: $85/hr

 

(G) UP facility: Cold sample shipping charges
Cost: $50 per package. (supplies added separately)

 

Users are expected to reimburse UP facility for all consumable supplies used.