Team:TU Delft-Leiden/Project/Life science/curli/cloning

From 2014.igem.org

Module Conductive Curli – Cloning

In the wet lab, we made constructs containing csgA and csgB, two of the genes involved in curli formation. Here you can find information with respect to cloning of the BioBricks for the Conductive Curli pathway.

Module Conductive Curli

Characterization

Inducible csgB, constitutive csgA/ csgA-HisTag

Final constructs

The final BioBricks are BBa_K1316013 and BBa_K1316014 (figures 1 and 2)

Figure 1: Plasmid containing the BBa_K1316013 BioBrick.

Figure 2: Plasmid containing the BBa_K1316014 BioBrick.

Cloning Scheme

For the construction of the final BioBricks BBa_K1316013 and BBa_K1316014, the two curli genes used (csgA and csgB) were obtained from the iGEM BioBrick BBa_K540000 from Lyon team 2011. In order to introduce the L-Rhamnose promoter upstream csgB and a constitutive Anderson promoter upstream csgA, the Golden Gate Assembly was used.

Golden Gate Assembly

To construct our BioBricks BBa_K1316013 and BBa_K1316014 three pieces of DNA had to be cloned into pSB1C3 . Both BioBricks encode an L-rhamnose promoter and the coding sequences of csgB and csgA or csgA-HIS. To work efficiently, we used the Golden Gate Assembly to construct our BioBricks. In this way we were able to restrict and ligate all the DNA fragments at once (see figures 3 and 4).

Therefore, three PCR fragments were generated, all of them properly designed so that, during the Golden Gate Assembly, the three fragments could only be assembled in the desired order and orientation.

Due to the small size of the constitutive Anderson promoter used ( BBa_J23110 ), the first half of the promoter was included at the 3' ending of the csgB reverse primer; and the second half of the promoter was included at the 5' ending of the csgA forward primer. As a consequence, the promoter would be properly reconstituted during the Assembly.

Note: the construct with CsgA carrying the 6xHisTag (BBa_K1316014) was made the same way as BBa_K1316013 but the reverse primer amplifying csgA was designed in order to incorporate the Tag right before the STOP codon of this gene.

Figure 4: Cloning scheme of the BBa_K1316013 and BBa_K1316014 BioBricks.

Inducible csgB and csgA

Final constructs

The final BioBrick is BBa_K1316015 (figure 5)

Figure 5: Plasmid containing the BBa_K1316015 BioBrick.

Cloning Scheme

For the construction of this BioBrick ( BBa_K1316015), both csgB and csgA genes were PCRed directly from the BBa_K540000 BioBrick from Lyon team 2011 and then placed behind the L-Rhamnose promoter of the iGEM BioBrick BBa_K914003.

Constitutive eGFP

Final constructs

The final BioBrick is BBa_K1316016 (figure 7)

Figure 7: Plasmid containing the BBa_K1316016 BioBrick.

Cloning Scheme

The BioBrick ( BBa_K1316016) was constructed amplifying the coding region of the Enhanced Green Fluorescent Protein (eGFP) from a comercial plasmid and placing it downstream the constitutive Anderson promoter ( BBa_J23110 ) on its Original iGEM plasmid.