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Andy Nguyen
Clostridum acetobutlylicum is a gram positve bacterium, which are really anaerobic for
growth conditions. Clostridium acetobutylicum has the potential to become economically
important since it produces energy rich biofuels. The Acetone-Butanol-Ethanol (ABE) process
was a common industrial practice in the early 1900s to make biofuels in cultures. The whole idea
was put off in the 1960s because it was not being competitive with oil cracking. Nowadays, with
increasing hunger for liquid fuels usable in transportation, alternatives to crude oil derived fuels
are being very intensively researching. Clostridium acetobutylicum is an ideal candidate for the
production of bio-synthetic fuel. The economical uses of Clostridium acetobutylicum are further
investigated by its ability to digest cellulose into fermentable sugars.
The researchers are trying to determined if the SMB_G1518 or SMB_G1519 gene
resulted an increased of butanol tolerance and to predict what would happened while
overexpressing the gene will do. The game plan of this experiment was to randomly approach
and rational design to determine butanol tolerant mutagens. Scientists chose this particular gene
for one reason because it has the same characteristics of the strain ATCC 824 to Clostridium
acetobutylicum. Used a program calle NCBI blast to create potential proteins to see what works
the best. The protein had a gene that was 30% similarity to the conserved butanol interacting
region in PKC. Find only one protein, which is the SMB_G1518, but the unusual part of the gene
was the location. The SMB_G1518 is located in a two-gene operon together with SMB_G1519,
so the stop codon of SMB_G1518 overlaps with start codon of SMB_G1519 indicating that both
the gene are some how linked.
The researchers used four different strains, which were Clostridium acetobutylicum
DC93 and DC94. The other were already in plasmid strain and were the 1731 (pImp1) and 1731
(p1518-1519). The plasmid uses in this study were pMTL009-1518, pMTL009-1519, and p1518-
Andy Nguyen
1519 the expression vector. The researchers used eleven specific primers. The Clostridium
acetobutylicum were grown anaerobically at 37 degrees Celsius in reinforced clostridial medium
for routine growth and making competent cells. Colonies were picked from agar-solidified plates
at least four days old and were heat shocked at 80 degrees Celsius for 10 minute before being
used to inoculate cultures. At all times, the growth in liquid medium was closely supervised by
using the 2802 spectrophotometer measuring the absorbance at 600 nm. The antibodies were
added at the final concentration at 100 ug/ml for ampicillin, 30 ug/ml for chloramphenicol and
50 ug/ml for erythromycin. All Clostridium acetbutylicum and E.coli strains were stored at -80
degrees Celsius in reinforced clostridial medium and liquid broth supplemented with 15%
glycerol to both.
According to figure 1A, the strain DDC14 had the highest absorbance reading, but the
strain, which the researcher was really after are the DC93 and DC94. Out of those strain, the
DC93 had a slightly higher reading than DC94, but had around the similar reading. According to
figure 1B, the growth was purposely put into 1% butanol growth condition. As expected the
DDC14 had the highest reading of all the strains. According to figure 2A, the growth conditions
were calibrated and the strain DSM 1731 had the lowest growth. According to figure 2B, the
strain 1731 (pImp1) and 1731 (p1518-1519) had similar growth pattern, but 1731 (pImp1)
yielded more growth. According to figure 4A, the data was tested to figure out the main
fermentation products after 50% growth inhibition degree was achieved and it seem butanol
yielded the most with the strain of DSM 1731 and DDC14 with 1% butanol growth. Acetate and
butyrate had below 4 g/L yields from various strains. Figure 4B, the data in this one was the
overexpressing the gene and indicates that butanol yielded more than the rest of the products
from DSM 1731 and DDC14 strains.
Andy Nguyen
The strain of DSM 1731 and DDC14 had the most effect on butanol growth, especially in
overexpressing. The (pImp1) and (p1518-1519) strain had the ability to withstand the toxicity
level of alcohol and gave the most butaol tolerance. The factors in the growth conditions were
entirely different from another, but under normal conditions the readings were high and under
1% butanol stress, the reading were low as expected because the bacteria are toxic to alcohol.
The take home message is that the two unknown genes of SMB_G1518 and SMB_G1519
leading to new discovery of what it does in Clostridium acetobuytlicum. Zinc finger protein was
found to be existed in many sequenced microbial strains and may have a chance to be involved
in alcohol like SMB_G1518 encoding protein. If so, it can be regared as potential target fro
engineering microbial alcohol tolerance.