E. coli cells refuse to grow! - (Mar/15/2012 )

Hi! I need help!

Hi,

I am a 2nd year graduate student in a structural biology lab. I have done the same prep many many times, only recently, it refuses to work no matter what I try! Here's what's been going on:

Background for prep:

I am recombinantly expressing a tetraspan membrane protein in E. coli (Rosetta Blue) cells growing in minimal media. I have been growing cells from the same glycerol stock for quite some time. One week, I did the growth/expression, and everything worked out fine. The next week, I did the growth/expression, and the cells grew to OD600 = 0.4 and then slowly started to die (OD reduced to 0.1 by the end of the day). I need them to reach 0.8 before induction with IPTG. This usually takes about 2 days growing at 20 degrees C.

Thinking that I must have forgotten a component in the minimal media or added too little glucose or something by mistake (I am typically very very meticulous but I was tired this week), I decided to grow again. The way I grow, I do a small starter in LB and then transfer a small amount of that culture to my large-scale cultures (I grow 150 ml in LB from a colony on a plate, spin down gently, resuspend in 40 ml fresh LB, and add 10 ml culture to each 1L miminal culture). The small scale cultures grew fine, but once I transferred to minial, kaput, no more growth. Now, I began to think it might be my media (since the culture was growing in LB). So, I remade all my stocks--glucose, antibiotics, CaCl, MgSO4, etc. and I grew again. Still, no growth happened in minimal, but cells did grow in the LB cultures.

Next, I decided it my be my glycerol stock...perhaps they were getting weak or something. So, I retransformed with my plasmid stock into RB cells. This worked fine, and I did a growth directly from this fresh transformation. Again, kaputt. Well, then I learned that our chloramphenicol was old and there was a new bottle. So, I made fresh CAP, and proceeded to repeat the growth, AGAIN. This time, my cells grew normally the first day (to 0.2) and the next morning, when I came in, they should have been around 0.6-0.8, but they were at 0.08-0.1. Dying cells.

My next plans of action is to (1) do a growth from a stock of cells from another guy in the lab. They are a different cell line and have a different protein (also a tetraspan membrane protein) but use the same media and antibiotics. The only difference is the growth is done at RT. This should tell me if something is up with my media. Then (2) I plan to resequence my plasmid. If it is fine, then I will (3) order a new stock of RB cells (although ours SHOULD BE fine).

I don't really know what else to do, and this problem is very puzzling to me because I have grown SO MANY times (probably 30-40) and never had a problem at ALL.

Thanks for reading and any advice,
K.

It's so weirrrrrrrrrrrrd ..I am new to lab too and keen to learn what happened there too, hope some lab masters coming out to help soon!!!

I'm confused.

How can your optical density reduce? Even if the cells are dead, they are still in the broth so your OD reading should at the very least remain the same.