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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.