Difference between revisions of "Part:BBa K525121"

Revision as of 17:28, 21 September 2011

S-layer cspB from Corynebacterium glutamicum with TAT-Sequence and lipid anchor, PT7 and RBS

Usage and Biology

S-layer proteins can be used as scaffold for nanobiotechnological applications and devices by e.g. fusing the S-layer's self-assembly domain to other functional protein domains. It is possible to coat surfaces and liposomes with S-layers. A big advantage of S-layers: after expressing in E. coli and purification, the nanobiotechnological system is cell-free. This enhances the biological security of a device.

This fluorescent S-layer fusion protein is used to characterize purification methods and the S-layer's ability to self-assemble on surfaces.

Important parameters

Experiment	Characteristic	Result
Expression (E. coli)	Localisation	cell membrane
	Compatibility	E. coli KRX
	Inductor for expression	L-rhamnose for induction of T7 polymerase
Characteristics	Molecular weight
	Theoretical pI

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1421
Illegal XhoI site found at 248
Illegal XhoI site found at 866
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1400
Illegal SapI site found at 647
Illegal SapI site found at 859
Illegal SapI site found at 1407

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 and the cell depris were still red. 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 proportionally high RFU in the detergent fractions and the MALDI-TOF analysis of the relevant size range in the polyacrylamid gel approved the insertion into the cell membrane (fig. 3).

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 % (v/v) sodium dodecyl sulfate; UTU = 7 M urea and 3 M thiourea; U = 10 M urea; NLS = 10 % (v/v) n-lauroyl sarcosine; CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

@@ Line 48: / Line 48: @@
 ===Identification and localisation===
-After cultivation 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 lysat was wahed with ddH<sub>2</sub>O. From the other part the periplasm was isolated using a osmotic shock. The existance of fluorescene in the periplasma fraction, shown in fig. 3, indicate that ''Brevibacterium flavum''-signal sequence is at least in part functional in ''E. coli'' KRX.
+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 ddH<sub>2</sub>O. 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 and the cell depris were still red. This indicated that the fusion protein intigrates because of the lipid anchor into the cell membrane. For testing this  assumption the washed lysate was treted with ionic, nonionic and zwitterionic detergents to release K525121 out of the membranes.
+The S-layer fusion protein could not be found in the polyacrylamide gel after a SDS-PAGE of the lysate and the cell depris were still red. 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 proportionally high RFU in the detergent fractions and the MALDI-TOF analysis of the relevant size range in the polyacrylamid gel approved the insertion into the cell membrane (fig. 3).