Research Thesis Assignment

EXTREMOPHILIC TEMPERATURE ADAPTATION IN LUCIFERASES; HIGH TEMPERATURE EXPRESSION SYSTEM

By

My Name

A Thesis Submitted in Partial Fulfillment of the Requirements For the Degree of Bachelor of Science in the Department of Biology

Claflin University

Orangeburg, South Carolina

March 2020

__________________________ ______________________________

Dr. So and So Dr. So and So

Thesis Advisor Committee Member

_________________________ ______________________________

Dr. So and So Dr. So and So

Committee Member Academic Advisor

Received by:___________________________________________________

Dr. Gloria McCutcheon, Chair, Department of Biology

Submitted to:__________________________________________________

Dr. Derrick Swinton, Dean, School of Natural Sciences and Mathematics

ACKNOWLEDGEMENTS

I would like to thank Dr. Panasik, my fellow lab mates, Dr. Harris, and everyone else within the Departments of Chemistry and Biology for their help in my thesis project.

THESIS STATEMENT

This research focuses on the use of Thermus thermophilus strain HB27 and the shuttle vector pMK18 as a high temperature expression system for expression of mutated bacterial luciferase from the psychrophile Vibrio harveyi at thermophilic temperatures to analyze temperature adaptations within the 8-stranded alpha/beta barrel protein fold.

ABSTRACT

DEPARTMENT OF BIOLOGY

JOHNNIE A. WALKER B.S. CLAFLIN UNIVERSITY, 2020

EXTREMOPHILIC TEMPERATURE ADAPTATION IN LUCIFERASES; HIGH TEMPERATURE EXPRESSION SYSTEM

Advisor: So and So

Proposal Dated: March, 2020

Proteins from psychrophilic, mesophilic, and thermophilic organisms have different properties that help them remain stable within their respective temperature ranges. Using the process of Directed Evolution – the creation of genetic diversity through error prone PCR and applying a selection for a desired phenotype, luciferase genes from a psychrophilic organism will be evolved to maintain stability at temperatures above the enzyme’s natural range. The evolved luciferase mutants will be analyzed to identify temperature adaptations within the protein fold. Using classical molecular biology techniques we are designing a shuttle vector containing the bacterial luciferase from the psychrophile Vibrio harveyi for expression and selection in both E. coli JM109(DE3) and Thermus thermophilus HB27. The shuttle vector chosen was pMK18 which contains origins of replication for E. coli and T. thermophilus and a thermostable kanamycin adenyl transferase gene. The optimal temperature for activity for the luciferase enzyme was determined to be 30°C. Growth rates for the high temperature expression strain were measured and optimal selection ranges were determined. The maximum temperature for expression and selection in E. coli was found to be 42°C and the selection and expression range in Thermus thermophilus was found to be 50°C to 70°C. Doubling times for HB27 in LB media with kanamycin were 2.0, 3.3, and 4.4 hours for 65°C, 60°C, and 55°C respectively. Doubling times for HB27 at 70°C and 50°C and a procedure for its efficient transformation will be conducted in the near future.

KEYWORDS AND ABBREVIATIONS

Keywords: thermostability, directed evolution, shuttle vector, origins of replication, doubling time, transformation

Abbreviations:

PCR polymerase chain reaction

OD600 optical density at 600 nm

LB Luria-Bertani Broth

HB27 Thermus thermophilus strain

SOB Super Optimal Broth

SOC Super Optimal Broth with catabolite repression

LIST OF TABLES

PAGE

Table 1. Results of HB27 growth on plates and doubling times 8

LIST OF FIGURES

PAGE

Figure 1. Cartoon of the eight stranded alpha/beta barrel motif 1

Figure 2. Crystal Structure of luciferase from Vibrio Harveyi. 1

Figure 3. Growth Curve of Bacteria 5

Figure 3. Growth Curve of HB27 at 55°C 8

Figure 4. Growth Curve of HB27 at 60°C 9

Figure 5. Growth Curve of HB27 at 65°C 9

TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS ii

THESIS STATEMENT iii

ABSTRACT iv

KEYWORDS AND ABBREVIATIONS v

LIST OF TABLES vi

LIST OF FIGURES vii

TABLE OF CONTENTS viii

INTRODUCTION 1

BACKGROUND AND LITERATURE REVIEW 2

MATERIALS AND METHODS 6

RESULTS 8

DISCUSSION & CONCLUSIONS 10

REFERENCES 11

INTRODUCTION

For many years researchers have attempted to understand structure-function relationships responsible for thermostability and the definition of an enzyme’s optimal and functional temperature range. While no general rules have emerged, research has focused on comparisons of mesophilic proteins with their extant thermophilic counterparts, which may have many amino acid differences, and rely on generalizations across different protein fold families. We propose that temperature adaptation may be “Fold Specific” – i.e. that the structural strategy employed to achieve thermostability may be specific to protein fold.

Figure 1. Cartoon of the alpha/beta barrel motif (Farber, 1990)

Figure 2. Crystal Structure of luciferase from Vibrio Harveyi. The enzyme consists of two barrel subunits (pdb file 1luc).

To obviate the need to compare across large amounts of genetic drift and to compare within one protein fold family, 8-stranded alpha/beta barrels (Fig. 1), we developed a methodology to take alpha/beta barrel psychrophilic enzymes (luciferase, Fig. 2) and evolve thermostable characteristics. Using “Directed Evolution”, mutating genes using error prone PCR and applying temperature selections, we endeavor to map structural “hot spots” for adaptation to temperature and compare them to a database of mesophilic and thermophilic alpha/beta barrel enzyme structures.

BACKGROUND AND LITERATURE REVIEW

Thermostability is not a well defined physiochemical property of proteins. The term is generally used to indicate the ability of a protein to function at higher temperatures for extended periods of time. If the protein can function at these higher temperatures, the protein remains folded in its native state. Many studies have claimed various factors that can increase thermostability in proteins. These factors include tighter packing of the hydrophobic core, electrostatic interactions such as ion pairs and hydrogen bonds, α-helix dipole stabilization, decreasing the conformational entropy or main chain flexibility of the unfolded state through the introduction of prolines, and the shortening of surface loops. It is known that not all of these factors have the same role in creating thermostability in members of all protein families (Leggio, 1999, Wallon 1997, and Fleming 1997).

The shortening of surface loops is considered a factor because evidence from molecular dynamic simulations states that long flexible loops unfold first during thermal denaturation and promotes unfolding in the rest of the protein. It has been seen in different studies that proteins from thermophilic organisms (those with optimal growth temperatures between 45°C to 85°C) have smaller, more compact loops when compared to similar counterparts from mesophilic organisms (those with optimal growth temperatures between 15°C and 45°C) (Leggio, 1999 and Wallon, 1997).

Decreasing the conformational entropy or main chain flexibility of the unfolded state with the introduction of prolines is also considered a factor in thermostability because studies have shown that site directed mutagenesis with prolines can increased thermostability of a protein. Proline is proposed to decrease the entropy of the unfolded state because its phi angle is restricted, thus decreasing conformational space. The entropy of the unfolded state determines the number of conformations a long polypeptide can obtain. The lower the entropy, the smaller the number of conformations that can be obtained by the polypeptide and the smaller the number of conformations, the greater the increase in rigidity with less fluctuations of the polypeptide backbone being allowed. It is proposed to decrease entropy with its introduction at the beginning of the N-terminus of α-helices, which stabilize helix dipoles. Helix dipoles refer to the overall charge of the N-terminus and C-terminus of helices. A study of 215 α-helices for amino acid preferences within a helix showed that the N-terminus is preferably negatively charged and the C-terminus preferably positively charged (Leggio, 1999 and Wallon, 1997).

At their respective optimum temperatures, thermophilic and mesophilic homologous proteins are thought to display similar degrees of flexibility and activity with the thermophilic proteins being less flexible at mesophilic temperatures (Song, 2000). The amino acid make-up of a protein determines its flexibility due to the constant fluctuation of the phi, psi, chi, and omega angles of the individual amino acids. So by increasing the number of residues that fluctuate less often like proline and others with bulky side chains, flexibility can be assumed to be lowered.

Ion pairs and hydrogen bonding are considered factors in thermostability because studies of certain protein families display higher numbers of ion pairs and hydrogen bonds in thermophilic proteins when compared to their mesophilic counterparts. Also studies have shown that “multiple ion pair networks” are more energetically favorable than just the sum of single ion pairs (Leggio, 1999 and Wallon, 1997).

Solvent accessible surface area describes both the hydrophobic packing of hydrophobic residues and the hydrophilic exposure of hydrophilic residues. Hydrophobic packing is considered a factor in thermostability because studies have shown that thermophilic proteins have smaller amounts of solvent accessible surface area in relation to the hydrophobic core and more hydrophilic residue exposure to solvent when compared to mesophilic counterparts. Also, cavities within the hydrophobic core of proteins have a destabilizing effect on stability. Thermophilic proteins maximize the favorable van der Waals interactions within the hydrophobic core when compared to mesophilic counterparts decreasing the unfavorable interactions with the solvent (Leggio, 1999 and Wallon, 1997).

The eight stranded α/β barrel protein fold (Fig. 1 in introduction), or TIM barrel (because it was first observed in triose phosphate isomerase, TIM) is one of the most common protein folding patterns in all known proteins. Most TIM barrels are enzymes; they include five of the six primary types of enzymes. There are nearly 900 TIM barrel structures within the CATH classification system and these barrels are amazingly diverse in sequence and function (Nagano, 2002 and Nagano, 2001). The archetypical α/β barrel motif consists of a β-strand followed by a loop connecting an α-helix, with this sequence being repeated eight times connected by seven β-turns. Enzymes with this motif often have helices, strands, and entire domains preceding, interrupting, and following the barrel (Farber, 1990). Also, the insertion of secondary structures usually occurs in the loop regions after a strand and preceding the canonical helix.

These previous studies have failed to establish characteristics that increase thermostability between individuals within protein families and folds, and those within diverse families. When doing these cross comparisons, individual amino acid changes that increased or decreased thermostability could not be observed by analyzing ion pairing, hydrogen bonding, solvent accessible surface area, and flexibility. Directed evolution comes into the fore front in understanding thermostability because it does allow individual amino acid changes to be tracked.

Directed evolution uses a random process in which error-prone PCR mutates a protein’s gene, creating a library of mutant genes. Once mutants are created, selection or high-throughput screening is used to identify the mutants containing the desired trait(s). Usually this process is repeated to enhance the desired trait(s). This technique has been widely applied in industrial processes and proves to be very valuable in improving and enzymes and creating new metabolic pathways (Chen, 2001).

As with this project, directed evolution can be used to study structure-function relations in enzymes by analyzing the biochemical and structural properties of desired mutants to understand the molecular basis of the observed new trait(s) in enzyme catalysis and/or stability (Lebbink, 1999).

Another important aspect of directed evolution is that there will usually be only one amino acid substitution per mutant generation. Thus changes in function can be attributed to the single substitution. When there is more than one mutation, functional change interpretation may not be readily apparent (Arnold, 1998). The information learned from directed evolution experiments could possibly be used to propose targets for rational site-directed mutagenesis (Chen, 2001).

Limitations with directed evolution are substrate specificity engineering, a narrow spectrum of mutation, and the need for a sensitive and efficient method to screen a large number of potential mutants. Engineering substrate specificity, a drastic change in enzyme function, would naturally need major changes to the polypeptide backbone to accommodate the new function. Directed evolution mainly improves a protein by point mutations with a bias for transitions (purine to purine, pyrimidine to pyrimidine) over transversions (purine to pyrimidine, vice versa) limiting the spectrum of mutation while nature has a broader spectrum with natural recombination (Chen, 2001).

A high temperature expression system is needed in this study of thermostability to express thermostable mutants at higher temperatures which E. coli cannot tolerate. This is why a thermophilic organism is needed in this study. Also, a shuttle vector containing origins of replication for two or more species is need to express mutants created from 42°C stable mutants expressed in mesophilic E. coli. And once a thermostable mutant is expressed in thermophilic T. thermophilus, the mutant can be harvested out of mesophilic E. coli which grow at less extreme and in easily manageable conditions.

Thermophiles are organisms that are at home in extreme environments susch as heating hay stacks, hot water lines, and hot springs. These organisms contain heat-stable proteins and protein synthesis systems. Special histone-like proteins keep DNA stabilized and the specialized cell membrane is made up of more saturated, more branched, and higher molecular weight lipids increasing the stability of the membrane (Willey, 2008).

The genus Thermus is located within one of the oldest phylogenetic branches of bacterial evolution with its species being some of the most extremely thermophilic bacteria known. Thermus spp. are aerobic, rod-shaped, nonsporulating, gram-negative, and rapid growers. The proteins produced by this genus of bacteria are stable at not only high temperature but also in organic solvents and high concentrations of urea and detergents. These bacteria and their proteins are highly applicable for biotechnological uses, such as their use in the functional selection of thermostable enzymes and the use of Taq polymerase in PCR (de Grado, 1999 and Koyama, 1986). Thermus thermophilus is a well studied species of Thermus and the strain HB27 is a commonly used strain in experiments needing a thermophilic organism.

Most Thermus spp. also contain plasmids that do not seem to give special advantages to the bacteria. These same plasmids have been used to create cloning vectors containing origins of replication for these thermophilic bacteria (de Grado, 1998). Using a previously designed thermostable kanamycin adenyl transferase, the shuttle vector pMK18 was designed using an origin of replication from a Thermus sp. ATCC27737’s plasmid and an origin of replication for E. coli from the plasmid pUC18 (de Grado, 1999).

Figure 3. Growth Curve of Bacteria (Tullmin, 2001).

After deciding to use HB27 as our high temperature organism, the determination of growth conditions and growth rates was needed to grow the cells efficiently at their extreme conditions. When growing liquid cultures of bacteria and creating a growth curve, there are four phases of the curve: lag, exponential (log), stationary, and death. The lag phase consists of cells being placed into fresh media and there is no immediate increase in cell number. The log phase consists of cells growing at their maximal rate possible, which is constant, for their environment with the cells doubling at regular intervals. The cells are usually uniform during the log phase which is why this phase is used in transformation protocols for maximum efficiency. The stationary phase consists of cells that have stopped growing due to nutrient starvation, toxic waste build-up, and cell population density. The death phase consists of lost cell population density due to possibly the lost of the ability to be cultured or programmed cell death (Willey, 2008).

Transformation is the introduction of recombinant DNA (in this case the shuttle vector) into competent cells. Competent cells are those that are readily capable of taking up foreign DNA. Certain species of bacteria have the ability to take up DNA with species being called naturally competent (Lodge, 2007). HB27 is considered to be naturally competent (Koyama, 1986). When cells are not naturally competent like Escherichia coli, cells are chemically treated within a chilled buffer containing divalent cations/basal salts to become competent before DNA is introduced or electroporation is used to make the cells take up DNA by sending a high voltage pulse through the bacterial suspension in a special cuvette (Lodge, 2007).

The high temperature expression system developed here will hopefully lead to the identification of structural “hot spots” for increased thermostability within the TIM barrel fold.

MATERIALS AND METHODS

MEDIA PREPARATION

Competent Thermus thermophilus HB27 and pMK18 were included in BIOTOOLS’ THERMOTOOLS Cloning Kit Ready to Use. The pMK18 shuttle vector contained a thermostable kanamycin adenyl transferase gene which allowed for selection of transformed cells with the antibiotic kanamycin. Kanamycin was always kept at a concentration of 10 mg/ml when used for selection. Luria-Bertani Broth (LB) was made by mixing 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 1 ml of 1M NaOH, and adding distilled water up until 1 L and then autoclaved. The trial media used for HB27 transformation (HB27 media) was made by mixing 8 g of tryptone, 4 g of yeast extract, 3 g of NaCl, 267 mg of sodium bicarbonate, 20-25 mg of calcium sulfate, 20-25 mg of magnesium sulfate, 20-25 mg of potassium chloride, and adding distilled water up until 1 L and then autoclaved. Super Optimal Broth (SOB) was made by mixing 20 g of 2.0% tryptone, 5 g of yeast extract, 0.58 g of 10 mM NaCl, 0.19 g of 2.5 mM KCl, and adding distilled water up until 1 L and then autoclaved. After being autoclaved, 1/100 volume of sterile filtered 1 M MgCl2 and 1 M MgSO4 was added to the SOB. Super Optimal Broth with catabolite repression (SOC) was made by adding 1/100 volume of sterile filtered 2 M glucose to SOB media. When making LB agar plates, 15 g of bacteriological agar was added before autoclaving. After autoclaving, the LB agar was slightly cooled and then poured into plates. A Tupperware container was purchased from Dollar General to retain moisture while growing HB27 at the extreme temperatures. A Beckman Coulter DU 800 Spectrophotometer was used for OD600 observations.

GROWTH ON AGAR PLATES. HB27 transformed with pMK18 was streaked onto LB agar/kanamycin plates, placed inside of the Tupperware container with a small beaker of water and wet paper towels (to retain moisture), and grown at 70°C in an incubator to observe growth. This was repeated at 65°C, 60°C, 55°C, 50°C, and 45°C.

GROWTH CURVES. A colony of HB27 transformed with pMK18 was selected from an agar plate and grown up overnight in a 3-ml liquid culture of LB and kanamycin to an OD600 of around 0.650. Then, 215 μl of the liquid culture was placed into a 250-ml Erlenmeyer flask containing 100 ml of LB and kanamycin. This mixture was placed into a shaking incubator at 65°C so that the OD600 of the culture, as time passed, could be observed. The OD600 of the culture was observed and recorded at one- to two-hour intervals until the OD600 of the culture peaked, which was usually greater than 1.00 (the cells’ environment becomes too dense inhibiting growth). Then, the optical density was graphed as a function of time to observe the exponential log phase and to calculate the doubling time for HB27 at 65°C. This was repeated for 60°C and 55°C.

CALCULATION OF DOUBLING TIMES. The OD600, N2, at a time, t2, that was closer to the end of the log phase and another OD600, N1, at a time, t1, that was closer to the beginning of the log phase were used to calculate the doubling time, Td (Willey, 2008).

Td = (t2 – t1) * _log(2)_

log(N2/N1)

TRANSFORMATION PROCEDURE A (Zymo Research Corp. Z-Competent E. coli Transformation Kit): First, a single colony of HB27 was taken from a LB plate, placed into a 15-ml Falcon tube filled with 3.5 ml of LB, and incubated at 70°C overnight to create a starter culture. The next day, 0.5 ml of the starter culture was used to inoculate 50 ml of SOB media in a 500 ml culture flask and shook at 200 rpm at 60°C until the OD600 was 0.4 – 0.6. Then, the culture was transferred to ice and shook for 10 minutes. After the ice incubation, the cells were pelleted by centrifugation at 3,500 rpm for 10 minutes at 4°C. Then, the supernatant was removed and the cells were re-suspended in 5 ml of ice-cold 1X Wash Buffer and re-pelleted. After re-pelleting, the supernatant was removed and the cells were re-suspended in 5 ml of ice-cold 1X Competent Buffer. Then, the cells were aliquoted by 200 μl into 1-ml microcentrifuge tubes and held at -80°C. The next day, 5 μl of shuttle vector pMK18 was added to 200 μl of thawed cells and incubated on ice for 15 minutes. Then, the 200 μl of transformation reaction was added to 800 μl of SOC media within a 15-ml Falcon tube, shook at 70°C for one hour with 200 μl being plated onto LB agar/kanamycin plates, and incubated overnight at 70°C.

TRANSFORAMTION PROCEDURE B: First, a single colony of HB27 was taken from a LB plate, placed into a 15-ml Falcon tube filled with 3.5 ml of HB27 media, and incubated at 70°C overnight to create a starter culture. The next day, 10 μl of the starter culture was placed into 3.5 ml of fresh, pre-warmed HB27 and the cells were allowed to grow to an OD600 of 0.4 – 0.8. Then, 1 ml of this culture is placed into a new Falcon tube with 5 μg of the shuttle vector pMK18, incubated at 70°C for 2 hours, plated onto LB agar/kanamycin plates, and incubated overnight at 70°C (de Grado, 1999 and Koyama, 1986).

TRANSFORMATION PROCEDURE C: First, a single colony of HB27 was taken from a LB plate, placed into a 15-ml Falcon tube filled with 3.5 ml of HB27 media, and incubated at 70°C overnight to create a starter culture. The next day, 10 μl of the starter culture was placed into 3.5 ml of fresh, pre-warmed HB27 and the cells were allowed to grow to an OD600 of 0.4 – 0.8. The culture was then aliquoted by 1 ml into 1.5-ml microcentrifuge tubes and frozen at -30°C overnight. The next day, 1 ml of this culture is placed into a new Falcon tube with 5 μg of the shuttle vector pMK18, incubated at 70°C for 2 hours, plated onto LB agar/kanamycin plates, and incubated overnight at 70°C (de Grado, 1999, Koyama, 1986, and Hanahan, 1996).

TRANSFORMATION PROCEDURE D: First, a single colony of HB27 was taken from a LB plate, placed into a 15-ml Falcon tube filled with 3.5 ml of HB27 media, and incubated at 70°C overnight to create a starter culture. The next day, 10 μl of the starter culture was placed into 3.5 ml of fresh, pre-warmed HB27 and the cells were allowed to grow to an OD600 of 0.4 – 0.8. The culture was then aliquoted by 1 ml into 1.5-ml microcentrifuge tubes and frozen at -30°C overnight. The next day, 1 ml of this culture is placed into a new Falcon tube and incubated at 70°C for 30 minutes. Then, 5 μg of the shuttle vector pMK18 was added to the 1 ml culture, incubated at 70°C for 2 hours, plated onto LB agar/kanamycin plates, and incubated overnight at 70°C (de Grado, 1999, Koyama, 1986, and Hanahan, 1996).

RESULTS

Table 1.

Temp.	Growth Observed	Doubling Time (hours)
70°C	Yes	Not Determined
65°	Yes	2.0
60°C	Yes	3.3
55°C	Yes	4.4
50°C	Yes	Not Determined
45°C	No	-

OD 600 vs. Time @ 55 Degrees Celsius

0

0.5

1

1.5

2

0 20 40

Time (hours)

Optical Density @

600 nm

Flask 1

Flask 2

Flask 3

Average

Figure 4. Growth Curve of HB27 at 55°C

OD 600 vs. Time @ 60 Degrees Celsius

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 20 40

Time (hours)

Optical Density @

600 nm

Flask 1

Flask 2

Average

Figure 5. Growth Curve of HB27 at 60°C

OD 600 vs. Time @ 65 Degrees Celsius

0

0.2

0.4

0.6

0.8

1

0102030

Time (hours)

Optical Density @

600 nm

Flask 1

Flask 2

Average

Figure 6. Growth Curve of HB27 at 65°C

GROWTH ON AGAR PLATES. When referring to Table 1, HB27 transformed with pMK18 displayed growth on the LB agar/kanamycin plates at 70°C, 65°C, 60°C, 55°C, and 50°C. No growth was displayed at 45°C.

GROWTH CURVES. When referring to Figure 5, the log phase at 65°C lasted from about 10 hours after the start of incubation to about 20 hours. When referring to Figure 4, the log phase at 60°C lasted from about 15 hours after the start of incubation to about 25 hours. When referring to Figure 3, the log phase at 55°C lasted from about 20 hours after the start of incubation to about 30 hours.

CALCULATION OF DOUBLING TIMES. When referring to Table 1, the doubling time of HB27 at 65°C is 2.0 hours. When referring to Table 1, the doubling time of HB27 at 60°C is 3.3 hours. When referring to Table 1, the doubling time of HB27 at 55°C is 4.4 hours.

TRANSFORMATION. The transformation protocols (A-D) did not produce cells transformed with pMK18. There was no growth on any of the LB agar/kanamycin plates.

DISCUSSION & CONCLUSION

As expected, the temperature range for HB27 growth was large extending from 70°C to 50°C. 50°C is the lowest temperature at which HB27 can be grown. The time for a culture of HB27 to reach the log phase of growth increased by 5 hours for every decrease of 5°C. The doubling time also increased by about one hour for every decrease of 5°C. The optical density did not peak around 1.0 because of the gradual loss of media due to evaporation allowing high optical densities to be observed; evaporated media was replaced as much as possible during growth. While trying to develop our own transformation procedure for HB27 to cut down on costs, the protocols developed so far have not produced transformed cells. The natural competency of HB27 was tested by Procedures B, C, and D. The cells were froze in Procedures C and D to test if freezing of the cells helped transformation by causing the formation of zones of adhesions like with E. coli (Hanahan, 1996). Even though transformation did not occur, HB27 is naturally competent so the correct protocol has just not been found by our group yet. Since E. coli and T. thermophilus are both gram-negative bacteria, Procedure A was tried to check a chemically competent procedure that worked with another gram-negative bacterium. The next step that will be taken will be to use electroporation for transforming HB27 due to the high efficiency established with electroporation (de Grado, 1999).

Drawing from the growth studies’ results, growth of Thermus thermophilus HB27 is directly proportional to temperature. The lowest temperature at which Thermus thermophilus can grow is at 50°C, which will be the lowest temperature at which selection can occur in HB27. E. coli has a maximum growth temperature of 42°C meaning once a mutant is highly stable at 42°C the next step would be to select at 50°C in HB27. Since natural and chemical competency procedures did not produce transformed HB27, electroporation will be tried next for transforming the HB27. In the future, growth curves and doubling times for 70°C and 50°C will be observed and calculated and the transformation protocol for HB27 will be developed to express thermostable mutants carried by the shuttle vector pMK18.

REFERENCES

Arnold, F. H. (1998) Design by Directed Evolution. Acc. Chem. Res. 31: 125-131.

Chen, R. (2001) Enzyme engineering: rational redesign versus directed evolution. TRENDS in Biotechnology. 19, No. 1: 13-14.

De Grado, M., Castan, P., and Berenguer, J. (1999) A High-Transformation-Efficiency Cloning Vector for Thermus thermophilus. Plasmid. 42: 241-245.

De Grado, M., Lasa, I., and Berenguer, J. (1998) Characterization of a plasmid replicative origin from an extreme thermophile. FEMS Microbiology Letters. 165: 51-57.

Farber, G. K. and Petsko, G. A. (1990) The evolution of α/β barrel enzymes. TIBS. 15: 228- 234.

Fleming, T. and Littlechild, J. (1997) Sequence and Structural Comparison of Thermophilic Phosphoglycerate Kinases with a Mesophilic Equivalent. Comp. Biochem. Physiol. 118A, No. 3: 439-451.

Hanahan, D. and Bloom, F. R. (1996) Mechanisms of DNA Transformation in Escherichia coli and Salmonella, Neidhardt, F.C. et al., eds. Cellular and Molecular Biology, ASM Press, Washington, DC.: 2448-2459.

Koyama, Y., Hoshino, T., Tomizuka, N., and Furukawa, K. (1986) Genetic Transformation of the Extreme Thermophile Thermus thermophilus and of Other Thermus spp. Journal of Bacteriology. April: 338-340.

Lebbink, J. H. G., Kaper, T., Bron, P., van der Oost, J., and de Vos, W. M. (1999) Improving Low-Temperature Catalysis in the Hyperthermostable Pyrococcus furiosus β-Glucosidase CelB by Directed Evolution. Biochemistry.

Leggio, L.L., Kalogiannis, S., Bhat, M. K., and Pickersgill, R. W. (1999) High Resolution Structure and Sequence of T. aurantiacus Xylanase I: Implications for the Evolution of Thermostability in Family 10 xylanases and Enzymes With βα-Barrel Architecture. PROTEINS: Structure, Function, and Genetics. 36: 295-306.

Lodge, J., Lund, P., and Minchin, S. (2007) Gene Cloning: Principles and Applications. Taylor and Francis Group: New York. pp. 42-44.

Nagano, N., Orengo, C. A., and Thornton, J. M. (2002) One Fold with Many Functions: The Evolutionary Relationships between TIM Barrel Families Based on their Sequences, Structures and Functions. J. Mol. Biol. 321: 741-765.

Nagano, N., Porter, C. T., and Thornton, J. M. (2001) The (βα)8 glycosidases: sequence and structure analysis suggest distant evolutionary relationships. Protein Engineering. 14, No. 11: 845-855.

Song, J. K. and Rhee, J. S. (2000) Simultaneous Enhancement of Thermostability and Catalytic Activity of Phospholipase A1 by Evolutionary Molecular Engineering. Applied and Environmental Microbiology. 66, No. 3: 890-894.

Tullmin, M. (2001) Bacterial Growth Curve. Water Pages Corrison-Club.com. http://www.corrosion-club.com/waterbactgrowth.htm .

Wallon, G., Kryger, G., Lovett, S. T., Oshima, T., Ringe, D., and Petsko, G. A. (1997) Crystal Structures of Escherichia coli and Salmonella typhimurium 3-Isopropylmalate Dehydrogenase and Comparison with their Thermophilic Counterpart from Thermus thermophilus. J. Mol. Biol. 266: 1016-1031.

Willey, J. M., Sherwood, L. M., and Woolverton, C. J. (2008) Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw Hill Higher Education: Boston. pp. 123-139.