For a better expression system of heterologous proteins in E. coli

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Hi. I am Daniel Guerra, professor and researcher at the Universidad Peruana Cayetano Heredia in Lima, Peru. I wish to introduce changes to BL21.

Which aspects of BL21(DE3) do you think need to be maintained and which ones should be improved?

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To know your degree of experience, how many different proteins (including mutations) have you expressed by recombinant technology? (include those you supervised and those that were not successful) *

One

Between 1 and 4

Between 1 and 10

Probably around 10

Definitely more than 10

Which of these E. coli strains have you used to produce proteins?

(If other, please type after "Otros...")

BL21(DE3)

BL21(DE3) pLys

Rosetta(DE3) [BL21 con pRARE]

BL21(DE3) SixPack

BLR(DE3)

Lemo21

None

Egyéb:

Kötelező

What are the difficulties you have faced?

(If other, please type after "Otros...")

Low expression of my protein

High expression but lost in insoluble fraction

High soluble expression but my protein is lost to degradation during purification

High soluble expression but my protein is inactive

Sometimes it expresses a good amount and other times it expresses very little, spontaneously

Using HisTag, I find many contaminants after elution

Using an affinity tag (not HisTag), I find many contaminants upon elution

Difficult growth before induction (it is slow or very limited)

The inducer (eg IPTG, arabinose, tetracycline) broke and had to be replaced

The toxicity of my protein of interest causes difficulties

It has been difficult to achieve a complete lysis

Egyéb:

Kötelező

Even knowing that alternatives must be tested for the expression of each protein, and that prior information on each particular protein helps to choose the route, we are not always systematic. In general, please indicate the degree of importance that you give to the following possibilities of variation:

It's the first thing to try

I would try it second

I would try it as a last resort

Not worth it

Different expression plasmids

Different strains of E. coli

Different culture media

Different concentrations of inducer (eg IPTG)

Different culture temperatures after induction

Different culture times after induction

Different tags (fusion, solubility or transport)

A eukaryotic expression system

Using a BL21(DE3) or one of its variants, to get good performance repeatedly, which of the following best matches your experience?

(If other, please type after "Otros...")

I can make passages in liquid without losing expression.

I can cryo-preserve my producer clone without losing expression.

I can keep the producing clone in a Petri dish for a couple of weeks without losing expression.

I have to do a transformation with fresh bacteria and plasmid each time.

Egyéb:

Kijelölés törlése

What qualities do you find desirable in a recombinant expression system in E. coli?

It's not important

It's a bit important

It is moderately important

It's very important

It is finely tunable for different amounts of expression

Produces zero background expression (leaking)

Production is not reduced after cryopreservation of the expressing bacterium.

The producing bacterium is compatible with the most used plasmids (eg pET, pACYC, pDest...)

The inducer is accessible and economical

The inducer is stable at room T°

It's not important

It's a bit important

It is moderately important

It's very important

It is finely tunable for different amounts of expression

Produces zero background expression (leaking)

Production is not reduced after cryopreservation of the expressing bacterium.

The producing bacterium is compatible with the most used plasmids (eg pET, pACYC, pDest...)

The inducer is accessible and economical

The inducer is stable at room T°

Please share any additional insights or experiences regarding difficulties with BL21(DE3) or another expression system in E. coli and aspects that would be desirable in a new prokaryotic system.

Thanks for helping! If you wish, type your name and email.

(Sharing this information is optional, but it helps even more).

Küldés

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