Construction
CLONING
The genes were ordered from IDT.The genes needed for our project were CtfA, CtfB and BdhB.
The pathway of conversion
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.
The sequences we ordered
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 in the 2015 Distribution.
Colony PCR after ligation in an empty vector. Longer products represent correctly inserted synthetic genes.
2. CLONING OF COMPOSITE PARTS
2.1. Ligation into vector with a double terminator
We ligated the genes from the pSB1C3 vector (step 1.1.) as a forward insert into the vecor pSB1C3 with a double terminator, provided in the 2015 Distribution (BBa_K823017).
Restriction of plasmids with genes as forward inserts and plasmid with double terminator as a forward vector. The inserts and the vector were isolated from the gel and ligated.
Colony PCR after ligation in a vector with a double terminator. Longer products represent amplified genes with double terminators.
Colony PCR after ligation in a vector with a double terminator. Longer products represent amplified genes with double terminators.
2.2. Ligation into a vector with promoter and RBS
We ligated each of the three genes with double terminator (step 2.1.) in a pSB1C3 vector as a back insert into the vector pSB1C3 with a strong promoter and a strong RBS, provided in the 2015 Distribution (BBa_K608002).
Restriction of plasmids with genes and double terminators as back inserts and plasmid promoter and RBS as a back vector. The inserts and the vector were isolated from the gel and ligated.
Colony PCR after ligation in a vector with promoter and RBS. Longer products represent combined vector inserts containing promoter, RBS, gene of interest and double terminator.
Colony PCR after ligation in a vector with promoter and RBS. Longer products represent combined vector inserts containing promoter, RBS, gene of interest and double terminator.
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.
SDS-PAGE of construct containing CtfA. The gene is expressed as evident by the band at 25 kDa in the lysate, but not in the control lysate.
SDS-PAGE of construct containing CtfB. The gene is expressed and mostly in the unsoluble fraction as evident by the band around 25 kDa in the lysate and the unsoluble fraction.
SDS-PAGE of construct containing BtfB. The gene is expressed and mostly in the unsoluable fraction as evident by the band around 35 kDa in the lysate and the unsoluble fraction.
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.
Membrane after Western blot of the construct containing CtfA. The gene is expressed and mostly in the unsoluable fraction, as evident by the stains.
Membrane after Western blot of the construct containing CtfB. The gene is expressed and partly in the soluable, partly in the unsoluable, as evident by the stains.
Membrane after Western blot of the construct containing BdhB. The gene is expressed and partly in the soluable, partly in the unsoluable, as evident by the stains. Lower molecular size bands are most likely partly digested proteins.