Testing Team Heidelbergs pSBX-vectors

The iGEM team Heidelberg constructed a set of expression vectors that contain a T7 promoter prior to the BioBrick prefix. This way RBS-CDS parts can be cloned into the vectors by standard BioBrick assembly and then expressed in BL21(DE3) by induction with IPTG.

Aachen Heidelberg vector Design.png
Heidelberg X-press vector design
The iGEM Team Heidelberg created new expression vectors with an integrated T7 Promoter

In collaboration with the iGEM Team Heidelberg the iGEM Team Aachen tested 8 of these vectors for their expression capabilities.

We successfully cloned and expressed one of our fusion proteins K139020 in a pSBX1A3 vector. You can read more about the background of this expression on the Galectin-3 page.

Attempting to Characterize the pSBX vectors

These are the tested constructs:

Plasmid Backbone Insert Length vector [bp] Length Insert [bp] Resistance
pSBX1A3-BBa_13507 pSBX1A3 (high copy) BBa_13507 3067 1226 50 mg/ml Ampicilin
pSBX1C3-BBa_13507 pSBX1C3 (high copy) BBa_13507 2982 1226 35 mg/ml Chloramphenicol
pSBX1K3-BBa_13507 pSBX1K3 (high copy) BBa_13507 3116 1226 50 mg/ml Kanamycin
pSBX1T3-BBa_13507 pSBX1T3 (high copy) BBa_13507 3322 1175 10 mg/ml Tetracyclin
pSBX4A5-BBa_13507 pSBX4A5 (low copy) BBa_13507 4307 1192 50 mg/ml Ampicilin
pSBX4C5-BBa_13507 pSBX4C5 (low copy) BBa_13507 4127 1186 15 mg/ml Chloramphenicol
pSBX4K5-BBa_13507 pSBX4K5 (low copy) BBa_13507 4331 1192 50 mg/ml Kanamycin

We also used an mRFP selection marker with a constant expression of mRFP as a positive control.

To find out more about the Results of the characterization check out the the collaboration page of Team Heidelberg.

Fluorescence data of pSBX cultivation
While the positive control did show an increase of fluorescence to a maximum of ca. 20000 relative fluorescence units, there appeared to be something wrong with the pSBX expression strains. We did not have the time to check what exactly was wrong with the cells, but it's not because of the vectors, because we successfully used the pSBX1A3 to express our K1319020 fusion protein.