Engineered Microbial Consortium for the Efficient Conversion of Biomass to Biofuels Page: V
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3.1.3 Substrates ............................................................................................. 40
3.1.4 Hydrolysis with Crude Cellulosome Preparation.................... 40
3.1.5 Protein Quantification of Cellulosome Preparations.................. 41
3.1.6 Reducing Sugar Quantification..............................................................41
3.1.7 Statistical A nalyses ................................................................................ 42
3.1.8 SD S-PA G E ........................................................................................... 4 2
3.1.9 Cloning, Construction and Expression ..................................................42
3.2 Cellulosome Capture: Expression of the cipC Gene of C. cellulolyticum............ 52
3.2.1 Amplicfication of pBBR1 MCS3.........................................................52
3.2.2 Amplification of the cipC Gene of C. cellulolyticum..................52
3.2.3 Amplification of N Terminal and C Terminal Sequences of the inaZ Gene
of Pseudom onas syringae ...................................................................... 52
3.2.4 Amplification of the Alkaline Phosphatase Gene (phoZ) of Streptococcus
pyogenes............................................................................................... . 53
3.2.5 Cloning and Expression of the Amplicons ...........................................53
3.3 Expected Results, Potential Obstacles and Alternative Plans................. 55
3.4 List of Combinations Tested for Ethanol Production.......................55
3.5 The Param eters M easured.................................................................................. 55
CHAPTER 4. RESULTS AND DISCUSSION.........................................................................56
4.1 SD S-PA G E ........................................................................................................... 56
4.2 G lucose Standard Curve .................................................................................... 57
4.3 Measurement of Endoglucanase Activity .........................................................58
4.4 Section C onclusions........................................................................................... 63
CHAPTER 5. DETERMINATION OF ETHANOL PRODUCTION USING RECOMBINANT
AND WILD TYPE Zymomonas mobilis ZM4 AND Clostridium cellulolyticum.............. 65
5.1 Ethanol Production from Cellobiose Using Recombinant ZM4 (pAA1) AND ZM4
(W ild T ype)...................................................................................................... . 7 6
5.2 Ethanol Production from Cellobiose and Microcrystalline Cellulose Using Z.
mobilis ZM4 pAA1, ZM4 WT and Clostridium cellulolyticum............... 78
CHAPTER 6. CON CLUSIONS ............................................................................................... 85
APPENDIX: SEQUENCE OF PLASMID pAAl....................................................................89
REFERENCES ............................................................................................................................. 97v
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Anieto, Ugochukwu Obiakornobi. Engineered Microbial Consortium for the Efficient Conversion of Biomass to Biofuels, dissertation, August 2014; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc699973/m1/7/: accessed July 17, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .