deletions | additions       diff --git a/Methods_Bill_Siena_this_is__.md b/Methods_Bill_Siena_this_is__.md  index 27ea99b..87ec977 100644  --- a/Methods_Bill_Siena_this_is__.md  +++ b/Methods_Bill_Siena_this_is__.md 
...
# Methods   [**Bill & Siena—this is all you guys!**] ## Protein production  ## Mutant production The BglB gene was codon-optimized for *E. coli* and cloned into a pET29b+ vector using Gibson assembly. Kunkel mutagenesis was used to generate mutations to BglB via the Transcriptic cloud laboratory platform. Sequence-verified plasmids were transformed into chemically-competent *E. coli* BLR cells. Single colonies from selection plates were inoculated into 5 mL cultures in 50 mL Falcon tubes and grown to saturation by shaking at 300 RPM for 24 hours at 37 degrees C in rich media (Terrific Broth, Fisher). Cells were pelleted by centrifugation and media was exchanged with Terrific Broth containing 1 mM IPTG. Following incubation at 18 C for 24 hours with shaking at 300 RPM, cells were lysed using BugBuster protein extraction reagent (AMD Millipore).  The BglB gene was codon-optimized for E. coli and cloned into a pET29b+ vector using Gibson assembly. Site-directed mutagenesis, utilizing the Kunkel method was used to generate mutations to BglB via the Transcriptic cloud laboratory platform. ## Protein purification  BglB mutant plasmids and E. Coli cells were incubated together on ice, allowing After centrifugation to clarify  the plasmid cell lysate, supernatant containing soluble proteins was transfered  to mix protein microcolumns containing 100 µL Ni-NTA resin (HisPur, Thermo). After washing  with E. Coli's surroundings. Heat shock at 42 degrees C and immediate incubation on ice resulted in E. Coli's uptake of the exogenous genetic material. The addition of terrific broth and incubation at 37 degrees C for an hour assisted 6 column volumes, proteins were eluted  in the recovery 200 µL  of E. Coli wash buffer containing 25 mM EDTA. Protein yield was asssed via A280  and the incorporation of the plasmid. The recovered cells were spread evenly on kanamycin resistant LB agar plates and were incubated at 37 degrees C overnight. Multiple colonies on the agar plates should be visible within 12 hours. confirmed using 4-12% gradient SDS-PAGE gels (Life Technologies).  Growth media, containing terrific broth and kanamycin solution, was created. A single colony was scraped from the incubated LB agar plates and dipped into the growth media in a falcon tube. The single colony contains the mutant plasmid, which contains a gene resistant to kanamycin. In this growth media, the transforming plasmid survives while the other bacteria are susceptible to kanamycin antibiotic. This process ensures that only transformed cells are able to grow. The falcon tube was covered and incubated with shaking for 37 degrees C for 24 hours. ## Activity assay using hydrolysis of pNPG  The previously incubated falcon tube was centrifuged and the resulting supernatant was dumped, leaving only the growth pellet activity  of the transformed cell. Induction media, containing terrific broth, kanamycin solution, and isopropyl β-D-1-thiogalactopyranoside (IPTG), proteins  was created.Induction media analyzed using a UV/vis spectrophotometric assay of p-nitrophenyl-β-D-glucosideas hydrolysis. Each mutant  was used to resuspend the growth pellet. IPTG induces assayed in technical triplicate. To determine  the expression thermal stability  of the target each  protein, by mimicking 50 µL aliquots of  the inducer allolactose technical replicates (with approximate concentration 0.1 mg/mL, 10-fold diluted from elutate) were incubated in a BioRad Thermal Cycler  in 96-well PCR plates using a gradient of 30 to 50 degrees C. Next, 25 µL of  the lac operon pathway. The falcon tube, thermal-cycled protein was transferred onto an assay plate  containing the resuspended growth pellet and the induction media, were covered and incubated with shaking 75 µL of 10 mM pNPG [**is this actually correct? Do we use 100 mM or 10 mM substrate  for 37 degrees C this assay?**]. Absorbance was monitored at 420 nm every minute  for 24 hours. 60 minutes and the rate of 4-nitrophenol production in M/min was calculated using a standard curve.  The expression cultures were lysed [Include notebook, master sheet,  and centrifuged in order to clarify the lysate. The proteins were then purified using immobilized metal ion chromatography. Absorbance of the proteins at 280 nm was quantified and further assessed using SDS-PAGE. assay data as CSV]  The activity of the proteins was analyzed using a UV/vis spectrophotometric assay of p-nitrophenyl-β-D-glucosideas hydrolysis. Using three columns per mutant, 50 ul of 10X diluted protein was thermo cycled for 30 min with an 8-step gradient from 30 C to 50 C. Next, 25 ul of the thermo cycled protein was transferred onto an assay plate containing 75 ul of 10X diluted pNPG. Absorbance was monitored at 420 nm every minute for 45-60 minutes and the rate of 4-nitrophenol production in M/min was calculated using a standard curve. ## Data analysis  [Assay procedure, short. Enough that an expert in Rates of 4NP formation at each of 8 temperatures from 30 C to 50 C were fit to  the field could reproduce logistic equation $$\textrm{rate} = frac{1}{1+e^{-k*(x-x0)}}$$ to determine the Tm-activity of each protein. Proteins  with some assumptions] a calculated Tm outside of our range of detection where discarded, leaving 119 unique samples.  [Include notebook, master sheet, and assay data as CSV] ## Rosetta modeling of $\DeltaG$   The Rosetta application `ddg_monomer` was used to calculate the predicted thermal stability of each point mutations (see attached Code S1 for the Rosetta methodology used).  [This is basically how we do it](http://www.bio.brandeis.edu/classes/biochem104/Protein_Melting_Curves.pdf), but we will want to write our own ## Machine learning?