Part:BBa_K525222

S-layer cspB from Corynebacterium halotolerans

S-layer Corynebacterium halotolerans

Important parameters

Experiment	Characteristic	Result
Expression (E. coli)	Localisation	Cell membrane
	Compatibility	E. coli KRX
	Induction of expression	L-rhamnose for induction of T7 polymerase
	Specific growth rate (un-/induced)	0.245 h^-1 / 0.109 h^-1
	Doubling time (un-/induced)	2.83 h / 6.33 h

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1103
Illegal XhoI site found at 559
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 226
Illegal NgoMIV site found at 1315
Illegal AgeI site found at 217
Illegal AgeI site found at 458
Illegal AgeI site found at 505
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 907
Illegal BsaI.rc site found at 214
Illegal BsaI.rc site found at 592
Illegal BsaI.rc site found at 994

Expression in E. coli

The CspB gen was fused with a monomeric RFP (BBa_E1010) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.

The CspB|mRFP fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase by supplementation of 0,1 % L-rhamnose using the [http://2011.igem.org/Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_S-layer_genes_in_E._coli autinduction protocol] from promega.

Figure 1: Growthcurve of E. coli KRX expressing the fusion protein of CspB and mRFP with and without induction. A curve depicting KRX wildtype is shown for comparsion.

Figure 2: RFU to OD₆₀₀ ratio of E. coli KRX expressing the fusion protein of CspB and mRFP with and without induction. A curve depicting KRX wildtype is shown for comparsion.

Identification and localisation

After a cultivation time of 18 h the CspB|mRFP fusion protein has to be localized in E. coli KRX. Therefor a part of the produced biomass was mechanically disrupted and the resulting lysate was wahed with ddH₂O. From the other part the periplasm was detached by using a osmotic shock. The existance of fluorescene in the periplasma fraction, showed in fig. 3, indicates that Brevibacterium flavum TAT-signal sequence is at least in part functional in E. coli KRX.

The S-layer fusion protein could not be found in the polyacrylamide gel after a SDS-PAGE of the lysate. This indicated that the fusion protein intigrates into the cell membrane with its lipid anchor. For testing this assumption the washed lysate was treted with ionic, nonionic and zwitterionic detergents to release the CspB|mRFP out of the membranes.

The existance of flourescence in the detergent fractions and the non-existancing fluorescence in the wash fraction confirm the hypothesis of an insertion into the cell membrane (fig. 3). An insertion of these S-layer proteins might stabilize the membrane structure and increase the stability of cells against mechanical and chemical treatment. A stabilization of E. coli expressing S-lyer proteins was discribed by Lederer et al., (2010).

An other important fact is, that there is actually mRFP fluorescence measurable in such high concentrated detergent solutions. The S-layer seems to stabilize the biologically active conformation of mRFP. The MALDI-TOF analysis of the relevant size range in the polyacrylamid gel approved the existance of the intact fusion protein in all detergent fractions (fig x).

In comparison with the mRFP fusion protein of K525121, wich has a lipid anchor, a minor relative fluorescence per OD₆₀₀ in all cultivation and detergent fractions was detected (fig. 3). Together with the decreasing RFU/OD₆₀₀ after 12 h of cultivation (fig. 2) indicates this rusult a postive effect of the lipid anchor on the protein stability.

Figure 3: Fluorescence pro OD₆₀₀ progression of the CspB/mRFP (BBa_E1010) fusion protein initiating with the cultivation fractions up to the detergent fractions of the seperate denaturations. Cultivations were carried out in autoinduction medium at 37 ˚C. The cells were mechanically disrupted and the resulting biomass was wahed with ddH₂O and resuspendet in the respective detergent. The used detergent acronyms stand for: SDS = 10 % sodium dodecyl sulfate; UTU = 7 M urea and 3 M thiourea; U = 10 M urea; NLS = 10 % n-lauroyl sarcosine; 2 % CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.