TITLE: Protein expression in Bacillus subtilis
AUTHORS: Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">B. subtilis </span><span>is a gram-positive bacteria used by both academia and industry as a protein production workhorse. This is due to its' their excellent fermentation properties, high production titers, and capacity to secrete proteins into the extracellular medium.</span></div><div class = "text-block"><span>This protocol describes how to express proteins in </span><span style = "font-style:italic;">B. subtilis.</span><span> The protocol is developed using KO7-S, although it might also work for other strains as well. The method is adapted from Rasmussen, M. D.; Bjoernvad, M. E.; Diers, I. Pectate Lyase Fusion for Expression and Secretion of Polypeptides. WO 00/75344, 2000 and Jensen, K.; Østergaard, P. R.; Wilting, R.; Lassen, S. F. Identification and Characterization of a Bacterial Glutamic Peptidase. </span><span style = "font-style:italic;">BMC Biochem.</span><span style = "font-weight:bold;">2010</span><span>, </span><span style = "font-style:italic;">11</span><span> (1), 47. https://doi.org/10.1186/1471-2091-11-47.</span></div></div>

[STEPS]
?. [Cal18-2 media preparations]
Prepare a stock solution of 2.0g/L Na2MoO4. Sterilize by filtration
?. [Cal18-2 media preparations]
Add a magnetic stirrer to the blue cap bottle and place the bottle on a stirring plate. Turn on the stirring, and make sure it's mixing well.
?. [Cal18-2 media preparations]
Add the following to the bluecap bottle:40g/L yeast extract1.3 g/L MgSO4·7H2O50 g/L maltodextrin (DE ~ 12)20 g/L NaH2PO4·2H2O6.7mL/L 2.0g/L Na2MoO4 stock solution6.7mL/L Trace metal solution100μL/L Pluronic L-61
?. [Cal18-2 media preparations]
Prepare a trace metal solution consisting of4.48g/L MnSO4·H2O3.33g/L FeCl3·6H2O0.625g/L CuSO4·5H2O7.12g/L ZnSO4·7H2OSterilize by filtration
?. [Cal18-2 media preparations]
Fill a blue cap bottle to ~80% of the desired final volume with MQ water.
?. [Cal18-2 media preparations]
Make sure that all of the ingredients are dissolved
?. [Cal18-2 media preparations]
Adjust to pH 6 with 5M NaOH
?. [Cal18-2 media preparations]
Add MQ water to the desired final volume
?. [Cal18-2 media preparations]
Sterilize by filtration
The media easily clogs filters, so choose a 0.45μM vacuum bottle top filter for this step and be prepared to use a few filters per liter
?. [Cal18-2 media preparations]
Store the media at  until needed
4 °C
?. [Overnight culture - Day 1]
Inoculate between  to  LB media with a single colony of the expression strain. Depending on the expression volume and overnight OD. The culture can be grown in in a 24-deepwell plate, a falcon tube or a shake flask
3 mL
50 mL
?. [Overnight culture - Day 1]
Grow the strain at
37 °C
Make sure to not incubate the overnight culture for longer than  . Using an overnight culture that has been incubating for longer than this, often results in non-reproducible results
?. [Expression - Day 2 - 4/5]
Prepare the desired volume of expression media in the desired vessel
Vessel and media volume has a massive influence on the final protein titers. In our experience, 24-deepwell plates in an incubator with a large shaking amplitude and CSTRs work well, while shake flasks give low protein yields. This is likely highly dependant on the equipment (e.g. incubators and the shape and baffle pattern of the shake flasks), and thus should be optimized for the individual labs
?. [Expression - Day 2 - 4/5]
Inoculate the expression media to an OD600 of 0.1
?. [Expression - Day 2 - 4/5]
Incubate the expression culture at  with 250 RPM shaking between  and
20 °C
The expression temperature and duration is dependant on the target protein, although the specified values seem to be a good starting point in our experience
?. [Harvesting - Day 4/5]
Harvest the culture by centrifuging at
Centrifuge: 6000 34, 5 min, 4 10
Depending on what the samples are for, the supernatant from the first centrifugation can additionally be centrifuged at   to clear out cell debris and other smaller contaminants
?. [Harvesting - Day 4/5]
Keep the sample  when working with it and at  for storage
on ice
-20 °C