Construction
Slovenia_HS_shema
CLONING
The genes were ordered from IDT .
The genes needed for our project were CtfA, CtfB and BdhB.
Slovenia_HS_X1
The genes needed for our project were CtfA, CtfB and BdhB.
Slovenia_HS_X1
CtfA and Ctfb are the genes coding the two polipeptide chains forming CtfAB (CoA-transferase) enzyme, that is in Clostridium acetobutylicum responsible for conversion of butyrate to butyryl-CoA. BdhB is the gene coding the butanol dehydrogenase enzyme, that is in Clostridium acetobutylicum responsible for conversion of butyryl-CoA to butanol.
All the DNA fragments ordered includes the CtfA, CtfB or BdhB gene from Clostridium acetobutylicum (NCBI: CAP0163) with a C-terminal His-tag, which has been codon optimized for expression in E. coli.
Slovenia_HS_X2
1. CLONING OF BASIC PARTS
1.1. Ligation into an empty pSB1C3 vectorWe first ligated the received gene fragments into an empty vector pSB1C3, provided by the 2015 Distribution.
Slovenia_HS_plasmid1
Slovenia_HS_X3
2. CLONING OF COMPOSITE PARTS
2.1. Ligation into vector with a double terminatorWe ligated the genes from the empty vector pSB1C3 as a forward insert into the vecor pSB1C3 with a double terminator, provided by the 2015 Distribution (BBa_K823017).
Slovenia_HS_plasmid4
Slovenia_HS_X4
Slovenia_HS_X5
Slovenia_HS_X6
2.2. Ligation into a vector with promoter and RBSWe ligated the gene with the double terminator in a pSB1C3 vector as a back insert into the vector pSB1C3 with a strong promoter and a strong RBS, provided by the 2015 Distribution (BBa_K608002).
Slovenia_HS_X7
Slovenia_HS_X7
Slovenia_HS_X8
Slovenia_HS_X9
3. PROTEIN EXPRESSION
Escherichia coli DH5α bacteria were transformed with the expression plasmids and grown in shaker flasks at 37 °C in LBC medium overnight. 1 ml samples were taken for analysis. Cells were collected by centrifugation at 6000g for 10 min and resuspended in 100 µl of H20. 10 µl samples were taken and 5 µl of SDS-PAGE loading buffer was added. Samples were cooked at 100 °C for 10 min and resolved on 12% SDS-PAGE gel.
Slovenia_HS_X10
Slovenia_HS_X11
Slovenia_HS_X12
Proteins were detected using Western blot as well. 12% SDS-PAGE gels were electroblotted for 1, 5 hours at 200 V, blocked in non-fat dry milk overnight and incubated the next day in two steps. A dilute solution of primary antibody was incubated with the membrane under gentle agitation for an hour. Then a dilute solution was incubated with the membrane under gentle agitation for an hour as well. The hydrogen peroxide method was for visualisation.