Editing NiCo21DE3 Cells (section)

=== Description:<br>  ===

<br>Chemically competent E. coli cells derived from BL21(DE3). Poly-histidine tagged recombinant proteins that are isolated by immobilized metal affinity chromatography (IMAC) are often contaminated with significant amounts of endogenous E. coli metal binding proteins. The protein expression strain NiCo21(DE3) has been engineered to minimize E. coli protein contamination of IMAC fractions: GlmS is mutated to eliminate binding to IMAC resins and three other proteins (SlyD, ArnA and Can) are tagged to enable rapid removal by chitin affinity chromatography.<br> 

*Superior alternative to BL21(DE3) for routine protein expression
*Improved purity of target proteins isolated by IMAC
*Identical growth characteristics as BL21(DE3)
*Deficient in proteases Lon and OmpT
*Resistant to phage T1 (fhuA2)<br>

<br> 

'''Compatible expression vectors/promoters'''<br>T7 or T7-lac promoter, Ptac, Ptrc, Plac, PlacUV5, ParaBAD vectors, PrhaBAD.<br>Note: Expression from vectors containing a T5-lacO promoter will not be regulated unless the expression vector also encodes the lacI gene or a compatible vector expresses LacI.<br> 

'''Transformation''' 

*Thaw a tube of NiCo21(DE3) Competent E. coli cells on ice for 10 minutes. 
*Add 1â5 µl containing 1 pgâ100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4â5 times to mix cells and DNA. Do not vortex. 
*Place the mixture on ice for 30 minutes. Do not mix. 
*Heat shock at exactly 42°C for exactly 10 seconds. Do not mix. 
*Place on ice for 5 minutes. Do not mix. 
*Pipette 950 µl of room temperature SOC into the mixture. 
*Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. 
*Warm selection plates to 37°C. 
*Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC. 
*Spread 50â100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 20â24 hours or at 25°C for 48 hours.<br><br>

'''Expression: '''<br> 

<br> 

*Transform expression plasmid into NiCo21(DE3). Plate on antibiotic selection plates and incubate overnight at 37°C. 
*Resuspend a single colony in liquid culture with antibiotic to produce a starter culture. Inoculate starter culture at a 1:100 dilution into expression media containing antibiotic. 
*Incubate at 37°C with shaking until OD600 reaches 0.4â0.8. 
*For most vector systems, induce expression with 40 or 400 µM IPTG for 3 hours at 37°C, 5 hours at 30°C or overnight at 16°C or 23°C.

<br> 

'''Removing&nbsp; IMAC Contaminating Proteins '''<br> 

E. coli SlyD, ArnA, and Can (carbonic anhydrase) are tagged with the chitin binding domain (CBD). Accordingly, these proteins may be removed by incubating the E. coli lysate or the IMAC elution fractions with chitin beads (NEB #S6651) or chitin magnetic beads (NEB #E8036). Binding of CBD-tagged proteins to chitin resin is compatible in a wide range of buffer conditions. Pooled IMAC fractions may be directly mixed with buffer-equilibrated chitin beads and incubated for 5â30 minutes to remove CBD-tagged contaminants from the His-tagged target protein. 

The following procedure is recommended:<br>Use 1 ml of chitin resin for each volume of lysate or IMAC pool corresponding to 1 gram of NiCo21(DE3) cell pellet. (or use 1 ml of chitin resin for every 100 ml of expression culture). Resuspend chitin slurry (stored in 20% ethanol) and transfer to a gravity flow column. Equilbrate chitin column with buffer similar or equivalent to the IMAC low imidazole buffer: (or use a buffer compatible with the downstream chromatography step). Seal bottom of chitin column and add cell lysate or IMAC fractions containing CBD-tagged contaminants. Seal top of column and mix by rocking for 5â30 minutes at 4°C. Elute void volume containing target protein by gravity flow and optionally add extra equilibration buffer to displace all buffer containing the target protein. 

(Alternatively, if using a mini-spin column incubate sample 5â30 minutes before centrifuging to elute the target protein). 

Analyze eluted protein by SDS-PAGE or Western blot to determine purity. Removal of CBD-tagged contaminants may be analyzed by Anti-CBD Monoclonal Antibody (NEB #E8034).<br><br> 

'''NEB Competent Cells'''<br> 

{| cellspacing="0" cellpadding="1" border="0" class="tableborder"
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| nowrap="nowrap" colspan="17" | Cloning Strain Properties
|-
| nowrap="nowrap" bgcolor="#c8d3a0" align="center" style="font-weight: bold;" | Strain Properties 
| bgcolor="#c8d3a0" style="font-weight: bold;" | NEB# 
| bgcolor="#c8d3a0" style="font-weight: bold;" | Transform<br> Efficiency<br> (cfu/µg)(1) 
| bgcolor="#c8d3a0" style="font-weight: bold;" | Strain 
| bgcolor="#c8d3a0" style="font-weight: bold;" | T1<br> Phage <br> Resistant 
| bgcolor="#c8d3a0" style="font-weight: bold;" | Library<br> Construc<br> -tion(2) 
| bgcolor="#c8d3a0" style="font-weight: bold;" | Cloning/<br> Sub<br> cloning 
| bgcolor="#c8d3a0" style="font-weight: bold;" | Blue/<br> White<br> Screening 
| bgcolor="#c8d3a0" | laclq 
| bgcolor="#c8d3a0" | lysY 
| bgcolor="#c8d3a0" style="font-weight: bold;" | Endo-<br> nucleaseI<br> Deficient(3) 
| bgcolor="#c8d3a0" style="font-weight: bold;" | Protease(4)<br> Deficient 
| bgcolor="#c8d3a0" style="font-weight: bold;" | FŽ 
| bgcolor="#c8d3a0" style="font-weight: bold;" | RecA¯ 
| bgcolor="#c8d3a0" style="font-weight: bold;" | T7 RNA<br> Polyme<br> -rase 
| bgcolor="#c8d3a0" style="font-weight: bold;" | Cytoplasmic<br> disulfide bond<br> formation(5) 
| bgcolor="#c8d3a0" style="font-weight: bold;" | Drug<br> Resistance(6)
|-
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| bgcolor="#d4d4d4" | str
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| C2925 
| 1-3x106 
| K12 
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| nowrap="nowrap" colspan="17" | Protein Expression Strain Properties
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| bgcolor="#d4d4d4" | nit
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| bgcolor="#d4d4d4" | C2566 
| bgcolor="#d4d4d4" | 0.6-1x109 
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| bgcolor="#d4d4d4" | â 
| bgcolor="#d4d4d4" | nit
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| bgcolor="#d4d4d4" | C3010 
| bgcolor="#d4d4d4" | 0.6-1x109 
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| bgcolor="#d4d4d4" | â 
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|-
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| C3013 
| 0.6-1x109 
| B 
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| cam, nit
|-
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| bgcolor="#d4d4d4" | C3022 
| bgcolor="#d4d4d4" | 0.6-1x109 
| bgcolor="#d4d4d4" | B 
| bgcolor="#d4d4d4" | â 
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| bgcolor="#d4d4d4" | nit
|-
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| C3025 
| 1 x 106 
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| bgcolor="#d4d4d4" | str, spec
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| C3027 
| 1 x 106 
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| bgcolor="#d4d4d4" | spec
|-
| <span style="background-color: navy; color: white;" />[http://www.neb.com/nebecomm/products/productC3029.asp SHuffle T7 Express] 
| C3029 
| 1 x 107 
| B 
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| spec(7)
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| bgcolor="#d4d4d4" | 1 x 107 
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| bgcolor="#d4d4d4" | cam, <br> spec(7)
|-
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| bgcolor="#d4d4d4" | 1-5x107 
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| <span style="background-color: navy; color: white;" /><span style="background-color: navy; color: white;" /> [http://www.neb.com/nebecomm/products/productC2528.asp Lemo21(DE3)]<br> 
| bgcolor="#d4d4d4" | C2528 
| bgcolor="#d4d4d4" | 1-3x107 
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(1) TE given are for high efficiency chemically competent strains. TE for electrocompetent strains are 1-4 x 1010cfu/mg. TE for subcloning strains are &gt;1 x 106.<br> (2) Restriction-deficient strain.<br> (3) Important for plasmid preparation.<br> (4) Lacks Lon and OmpT protease activity.<br> (5) Constitutively expresses a chromosomal copy of the disulfide bond isomerase DsbC.<br> (6) nit = nitrofurantoin, tet = tetracycline, cam = chloramphenicol, str = streptomycin, spec = spectinomycin<br> (7) Resistance to low levels of streptomycin may be observed. 

<br>

'''SHuffle® strains''' from NEB are engineered E. coli strains capable of expressing proteins with increasing disulfide bond complexity in the cytoplasm. SHuffle strains express the disulfide bond isomerase DsbC within the cytoplasm. DsbC isomerizes mis-oxidized substrates into their correctly folded state greatly enhancing the fidelity of disulfide bond formation. Cytoplasmic expression also results in significantly higher protein yields of disulfide bonded proteins when compared to periplasmic expression. SHuffle strains are sensitive to kan, amp, tet and in most cases, cam, which makes them able to express proteins from a wide variety of expression vectors offering greater versatility in experimental design. <br>

<br>

'''Lemo21(DE3)''' offers the host features of BL21(DE3) while also allowing for tunable expression of difficult clones. Tunable expression is achieved by varying the level of lysozyme (lysY), the natural inhibitor of T7 RNA polymerase. The level of lysozyme is modulated by adding L-rhamnose to the expression culture at levels from zero to 2000 µM. When Lemo21(DE3) is grown without rhamnose, the strain performs the same as a pLysS containing strain. However, optional addition of rhamnose tunes the expression of the protein of interest. For difficult soluble proteins, tuning the expression level may also result in more soluble, properly folded protein. <br>

<br>

[[Category:Protocols]]