IGEM team meeting notes 3/31/14

iGEM planning 3/31/14 (copied from Ashley’s notes, https://www.evernote.com/shard/s68/sh/0d4b54dd-80df-4481-b4f4-078279c4eb38/f3272686cb3b7a3876ff8887e2d962a1)

What makes yeast competent? e.g. http://www.bio-protocol.org/wenzhang.aspx?id=96

Background:

milk optimized to have as much fats, calcium, liquids as possible at the same time while still remaining liquid; in stomach turns into a gel that sits there, giving nutrients time to get into body

~dozen proteins in milk

whey vs casein protein

cow’s milk, 80% casein & 20% whey

alpha s1 ~ 32% alpha s2 ~ 8% beta ~ 32% kappa ~ 8% but K is what’s responsible for coagulation (turning into gel in stomach)

all hydrophobic so do not want to stay in soln in H2O

Ca2+ tends to precipitate out proteins

K casein: AA 169

N terminus is the hydrophobic end, C terminus is soluble part

hydrophilic tail has negative charges

Histidine residues + Nickel cation…..create a fake micelle (Craig)

drop in pH (naturally occurs in stomach)…burst of H+, cancels negative charges, collapses tails

loss of k casein results in hydrophobic micelles which cluster together and become complex micelle structures capable of capturing fat globules

need to read up more on the role of Calcium

two micelles sticking together involves irreversible changes….interactions between different casein proteins

calcium phosphate inside micelle

recombinant chymosin (Marc has purified chymosin already)

https://microbewiki.kenyon.edu/index.php/Microbial_production_of_recombinant_chymosin

sinerisis: how much liquid a gel weeps out

rennet: coagulation (protein degrading) enzymes; derived from ruminant stomachs

    review proteinase, protease, proteolytic enzymes
    all members of aspartic proteinase

after cleaving, N-terminal side is para-K casein

CMP is soluble, does not get coagulated into cheese, gets processed out with whey proteins

soluble tail from k-casein - possible to attach it….. tricks to be able to maximize protein production? other types of casing co-expressed with K-casein to form micelles

AA 105-106 (very vulnerable to aspartic proteinases) hydrophilic tail ends @ AA 169

Do micelles form spontaneously or are they dependent on milk ducts?

micelles are 200 nm - 2 um

Plan of attack:

one group replicating Kim paper and putting that in BioBrick format (this alone is sufficient for iGEM project)

another group try and express full-length K casein (more difficult due to hydrophobic head)

third group working on other 3 types of caseins to see if they can be expressed at all; at the very least make the bio bricks for these proteins

possible fourth group: modification of lipid biosynthesis pathways in yeast (butter in yeast?)

Marc: take existing vegan oil (like palm oil) and see if we can fully saturate it

Craig: increase # of micelles per unit area

Marc: nutritional yeast

Patrik: bacterial modifications to cheese

possible sponsor: DNA 2.0 (***call Amanda!!) —> add to list of sponsors!!

Craig: plant cells (or plant oil) to use as

Discussion about interest in plant project…most interest in cheese project

    DNA gun from glowing plant project
    Patrik: main hurdle is that there’s no plant expert

enzymatic vs. nonenzymatic stages of coagulation

Cheese making day!

Cheese making fundraiser; cheese making classes @ workshop weekends

    acid treated cheese….basically ricotta

homogenization more of an issue than pasteurization

Marc made a cheese press: container with holes in the sides (all food safe); a piece of plastic the same size; also a hinge that’s these same size;

Patrik: easiest way to make cheese is to put yogurt in a plastic bag and let it sit/hang over night

lactic acid bacteria:

next steps:

me: add DNA 2.0 to potential sponsor list; talk to Amanda about who to contact there

cheese making day: start planning what could be made in relation to project goals

cheese making fundraiser, cheese trail, Marin

learn more about genetic engineering of yeast: shuttle vectors, etc.

involvement of DIY molecular cloning class