Part:BBa_K771000

Membrane Anchor 0:ssDsbA-Bla-LGT

SsDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. SsDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the N-terminus, engineered membrane proteins are expected to be anchored onto inner membrane of E.coli .Lgt is an inner membrane protein of E.coli with seven transmembrane segments and has been successfully over expressed in E. coli without causing harm to cells.

Fig.1:Details of fusion protein BlaLG for membrane localization

To justify the localization ability of fusion membrane proteins mentioned above, GFP is fused to the C-terminus of Lgt[BBa_K771401]. The fusion protein in under control of araBAD promoter.

Fluorescence Test

To visualize the localization of BlaLG fusion protein, we adopted fluorescence test. GFP fused to the C terminus of BlaLG enables us to have a closer look at where the membrane proteins are localized . Under laser confocal microscope, we can observe the location of the fluorescence, thus to confirm the exact subcellular localization of the fusion protein.

Fig.2Bacteria carrying BlaLG induced in different concentration of L-arabinose.

It is observed that green fluorescence intensity of E.coli margin is higher than that of cytoplasm. Fig.2 proves that BlaLG has been localized to membrane.

Antibiotics Test

In this section, we test the membrane localization ability of fusion membrane protein by observing host cells' growth phenotype under different level of Ampicillin. Besides, we find the optimal inducer (L-arabinose) concentration for expressing membrane proteins.

β-lactamase，a bacterial enzyme which must be exported to the periplasm in order to confer significant resistance to β-lactam antibiotics, such as ampicillin. Note that N-terminus of Lgt faces the periplasm and C-terminus faces the cytoplasm . Hence, if our fusion membrane protein is correctly anchored to membrane, β-lactamase is expected to be functional and host cells should be able to grow on culture media containing ampicillin.

Fig.3:Growth phenotypes of E. coli cells expressing BlaLG on LB agar media with concentration of inducer L-arabinose from 0 to 0.2%. We increased the concentration of ampicillin from 0 to 200 (μg/ ml).Bacteria carrying BlaLG were able to grow at ampicillin concentration of 200 μg/ ml with induction

Fig.3 showed that our fusion protein has been correctly localized to membrane. Besides, a very low L-arabinose concentration at 0.02% is already enough to induce sufficient amount of membrane protein.

To further characterize features of the Membrane Scaffold System and find optimal inducer concentration, we test the growth condition of E.coli carrying gene of BlaLG under different concentration of L-Arabinose and Ampicillin. We expect to see more colonies grown at higher Ampicillin concentration if the inducer concentration is optimal.

Fig.4:Growth condition of E. coli cells expressing BlaLG on LB agar media with concentration of L-arabinose and Ampicillin. Single colony is picked and cultivated at 37℃ until OD value reaches 0.7. Bacteria cultures are diluted by 1:100000 and coated onto plates containing different concentration of L-Arabinose and Ampicillin. Growth condition is measured through counting colonies on plates.

The result apparently showed that at L-Arabinose concentration of 0.1% and 0.2%, more bacteria could grow on high concentration of Ampicllin. Thus, L-Arabinose concentration of 0.1% and 0.2% best suits membrane protein expression in Project Membrane Magic.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1747
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 794
  Illegal BsaI site found at 1368