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Tips on fermenting DH5alpha and MG1655


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#1 linli

linli

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Posted 23 September 2010 - 06:09 AM

Hi,
I知 trying to ferment E.coli cells and I知 new in the area of fermentation so I hope somebody can help me.

I知 doing fermentation in a 2L fermenter. I have used both DH5alhpa and MG1655.

My fermenter charge (media) is this:
Ingredients Concentration Amount (mg)
Potassium phosphate monobasic 6.8 g/L
Magnesium sulfate hepta hydrate 2.3g/L
Trace Minerals stock solution 1% V/V
Sigma 204 antifoam 0.012% V/V
Water - Up to 66% of working volume


Trace mineral stock solution:
Ingredients Concentration (g/L)
Ferric citrate 6
Ethylene diamine tetraacetic acid 1.6
Manganese chloride (tetrahydrate) 1.5
Zinc acetate dihydrate 0.8
Boric acid 0.3
Sodium molybdate dihydrate 0.25
Cobaltous chloride hexahydrate 0.25
Cupric chloride 0.118


To my fermenter charge I also add 21.2g/L glucose (50%w/w). And pH adjustment is done with 20% ammonia.

Is it something else I need to add to the media for good growth? Read somewhere that DH5alpha need arginin as supplement in mineral media, do I need that? I also read that the growth rate of MG1655 is 15% higher with addition of pyrimidine, do I need to add that?

When I fermented with DH5alpha the cells grew pretty good but now when I use exactly the same protocol with MG1655 the cells does not starts to grow. When I add the preculture of MG1655 into the fermenter, they immediately starts to consume oxygen but at a low level. The level of oxygen consume are constant for about 6 hours and then the consume of oxygen stops. For me this indicated that the cells are alive during the first time but never starts to divide, and after 6h they die. I have done the fermentation three times now with the same results. Does somebody have an idea why DH5a grow while MG1655 doesn稚? What can be the reason why the cells never get out from the lag phase?

Preculture is done in modified LB, see protocol below. Is it any special reason to use this instead of LB (I just had it in my protocol)?

Table 1: Modified LB medium for seed culture
Ingredients Concentration
Sodium Chloride 10 g/L
Yeast Extract 10 g/L
Ampicillin 100オg/ml
Water -


Some times I have concentrated the preculture by centrifugation at 3000xg, 22˚C for 15 min and resuspended in fermenter charge, can that hurt the cells? Is it better to cool the cells before centrifugation?

I知 very grateful for tips, since I have run out of my own ideas.

Thanks Lina

#2 protolder

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Posted 23 September 2010 - 10:29 PM

Hola Lina I donエt know what you want produce, but you are using DH5 that is a clonnig strain. Neither I donエt understand the use of minimal medium but you have your reasons. Revising genotype of strains you have to add thiamine and glutamin whose the strain is defficient(DH5) and MG1655 needs rifoflavin. Probably the first part of grown is due to the components of preinoculum,but you need a nitrogen source in the medium formula. 1g/l of glucose is enought to support the grown untill 4-5 ud OD 600nm, increasing it in bacteria gets to incompltete oxidation forming acetic acid (as yeast produce alcohol aerobically) and compromising the viability of culture. Before charge a fermenter, make some assays of media in flask to find the best condition of cultures. Good luck




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