Re: [DIYbio] Re: Exocytosis in Bacteria

In the study you linked the authors mention exactly the reason why I want to exocytose: "Another advantage is that correct formation of disulfide bonds can be facilitated because the periplasmic space provides a more oxidative environment than the cytoplasm." I want to secrete the exoskeletal proteins I'm working with for the iGEM project I mentioned earlier. The problem with having them remain inside the cell rather than being secreted is two-fold: one, because the inside of the cell is generally reducing, equilibrium is working against maximum crosslinking of the proteins by disulfide linkage, and two, because protein aggregation inside the cell means crosslinking will be disproportionately dense where cells used to be.
I'm going to attempt the type II pathway, just because it's the most commonly used. I'll report back if I get anything working.

As for protein design, I can personally attest to how difficult it is. We've spent months in the lab I work in just trying to get parameters right for stochastic simulations of protein folding. And if the protein requires any kind of covalent interaction with a ligand for folding, you have to use QM models, which are still extremely computationally heavy. Artificial selection seems like the best way to get a protein to optimize for the time being.

On Sunday, February 8, 2015 at 4:27:20 PM UTC-8, Josiah Zayner wrote:

Jacob:
I am not saying it won't work. I am not saying I don't support any work you do, you know I do lots! What I am more saying is what kind of throughput do you want? What do you mean by useful? What problem are you trying to solve? Why are you trying to exocytose?

Overexpressing any protein places a metabolic strain on the cell. Some more than others. It is extremely difficult to understand why some proteins express easily at 100mg/L and other struggle for 1mg/L. I have worked with graduate students who spent months.years attempting to optimize the expression of a single protein.

Here is a olderish (2004) review on secretion systems usage in protein overexpression: https://drive.google.com/file/d/0B_R75gIJvkFUWVdjQU9aNnU3bVU/view?usp=sharing

Nathan:

>I understand you're saying there's a burden from a 'natural'
>bacteria-pulled-from-its-

>habitat perspective, but it seems ridiculous
>when compared to how humans have transformed organisms with
>agriculture. If the bacteria aren't doing what you want, the you
>haven't engineered it properly. Thermodynamics, metabolism, entropy
>are all things to think about, but the earth doesn't have a 'problem'
>per-se... we're close to the sun, cold and entropy increases with the
>distance from the sun, so we've got the energy.

Hmm, I don't know what you mean by the Earth not having a problem?

Proteins are difficult to engineer. If Scientists knew how to engineer proteins and metabolic pathways "properly" we probably wouldn't be having this discussion. Not even getting into how things are regulated on the DNA and RNA level, protein complexity is insane orders of magnitude beyond what we can test. Current best methods can look at 10^10-10^15 sequences. This means that one cannot even sample the entire sequence space for a 15 amino acid protein. Sure we can find local energy minima or local rate maxima in protein function optimization but I might be as brash to say that no protein we know of has ever achieved a global energy minima or rate maxima in function. There is a huge difference between what is possible and what is probable.

On a minorly related stream of thought, one question that interests me in my work is "What kind of sequence space have bacteria actually explored?" Anyone ever read a paper on this?

JZ

On Sun, Feb 8, 2015 at 1:34 PM, Jacob Palumbo <palumb...@gmail.com> wrote:

Slapping on a signal peptide is definitely something that could be done. But low throughput is a problem. Random autolysis could also serve the same purpose, as entire colonies would generally survive while individuals wouldn't necessarily.

Do you think overexpression of proteins tagged for secretion would place too much of a metabolic strain on the cell? Even with lower throughput, overexpressing a protein enough may get enough to secrete to be useful. Of course, that still doesn't solve the problem of ligands, as you mentioned.

Jake

On Friday, February 6, 2015 at 11:22:22 AM UTC-8, Josiah Zayner wrote:

The problem with this Jacob is that you are fighting against thermodynamics and metabolism. The amount of energy cost to export proteins is huge, in the folding, transport, generation of transport proteins. Also, one needs to think about ligands, which many proteins contain or need to fold. I think the reason that this avenue has not been explored thoroughly is that you will be trying to optimize many different processes with many bottlenecks. Also, disrupting cells using some detergent (SDS or something) and maybe some freeze-thawing is darn easy. It would probably be extremely difficult to match normal expression methods for the wide variety of proteins that people want to purify. This could be useful for some niche cases but usually that just involves slapping a signal peptide on the proteint.


Josiah

On Thursday, February 5, 2015 at 2:40:18 PM UTC-8, Jacob Palumbo wrote:

Hey all,

I'm working on a project where it would be extremely useful to have bacteria (E. Coli specifically) exocytose proteins continuously, rather than grow a culture and lyse it the old fashion way. I've found a few papers on this so far (1, 2, 3), but they are all for Gram-positive bacteria, which E. Coli is not. If need be, I could switch over, but it would be nice to not have to. I do have some information the Sec pathway in Gram-negative bacteria, but it is somewhat general.

Anyone have an experience/insight into this?

Thanks,
Jake

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