Part:BBa_K823049

P_cotYZ - cotZ (-2aa) - gfp - Terminator

This composite part expresses a GFP fusion protein on B. subtilis spores.

The LMU-Munich 2012 team aimed to display proteins on spores for usage as Sporobeads.

For creating fusion proteins on Bacillus spores, the crust protein cotZ -2aa were brought into Freiburg Standard for in-frame translational fusions and put under the control of the native promoter P_cotYZ.

Evaluation

We evaluated five constructs in two different Bacillus strains (W168 and W168∆cotZ)

All the Sporobeads were investigated by fluorescence microscopy and analysed with ImageJ and the statistical software R. The intensity bar charts show the fluorescence intensity, while the 3D graphs illustrate the fluorescence intensity spread across the spore surface, which correlates with the distribution of our fusion proteins. For analysis we measured the fluorescence intensity of an area of 750px per spore by using ImageJ and evaluated it with the statistical software R. The following graph shows the results of microscopy and ImageJ analysis.

It appears the strain with the construct P_cotYZ-cotZ_-2aa-gfp-terminator in the ∆cotZ background had the strongest fluorescence. Thus, this would be the strain for future Sporobeads with special functions. In some 3D graphs the picked cell does not respresent the average fluorescence intensity depicted in the bar chart because it was chosen randomly.

NOTE: There are two versions of the cotZ gene in the registry, cotZ -2aa (used for this device), and cotZ. For our purpose, cotZ -2aa worked better.

For creating fusion proteins for the Sporobeads, the genes gfp, cotZ and cgeA were brought into Freiburg Standard whereas we created two different versions of the crust proteins. The restriction site NgoMIV was inserted just after the startcodon of the gene of the crust protein. Since this restriction site adds six additional basepairs, the resulting gene is two codons longer than the native cotZ. It was not know if this insertion has any effect on protein expression. Therefore we created two versions, this one, where we deleted two six basepairs, cotZ -2aa, and another, "cotZ" that is 2 aa longer.

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 237
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 835