I am providing you with all of the articles that you should include in this thesis. This is a big assignment. Do Not Rush It. The template has been provided. I need this by March 11th. As time goes on, I will add more literature reviews for you to add in. 

For the results section, since I have not done actual research use the results from the articles and say how you would do it. 

If you have any questions, ask me. 

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 Uni

v

ersity

Orangeburg, South Carolina

March

2

0

20

__________________________
______________________________

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 pMK

1

8 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,

0.5

8 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

0.6

50. 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

No

t 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.

v

Biol 491 Senior Seminar

Carmen Major

October 16, 2020

Ethnicity is associated with alterations in oxytocin relationships to pain sensitivity in women

It is known that African Americans experience greater pain associated with many different clinical conditions as well as a greater sensitivity to experimental pain tasks when compared to non- Hispanic whites. Not only do African Americans show reduced pain tolerance to experimental pian tests but there is also an absence of the expected endogenous pain inhibitory mechanisms that is documented in Caucasians. Only Caucasians show the expected positive association between higher pain tolerance and greater blood pressure, plasma norepinephrine, and cortisol. All correlations involving these biological measures were very low, which suggests there is inherent differences, alterations or adaptions in one or more biological pathways involves in pain inhibition that could possibly contribute to the ethnic differences in pain. The purpose of this article was to examine the relationship between plasma oxytocin (OT) and pain sensitivity. This study also explores the relation between plasma oxytocin and other factors that are known to influence pain perception.

The association of plasma oxytocin concentration was tested with three types of experimental pain: Ischemic pain tolerance, Hand cold pressor task and Thermal heat pain task. To test this theory, 48 women, 25 African American and 23 non-Hispanic White, were used to examine the effects of ethnicity, gender, and stress on physiological mechanisms involved in endogenous pain regulation. These women were healthy non-smokers, pre-menopausal, medically healthy, normotensive or pre-hypertensive and free of chronic disease or pain disorders. Additionally, none of them took prescription medication of any kind. Each woman underwent an identical experimental laboratory testing once in each of the three phases of the menstrual cycle (early follicular, late follicular, and luteal phases). Only results exclusive to late follicular phase, days 8-12, testing were reported in this article.

During the ischemic pain procedure, the submaximal effort tourniquet was used to occlude blood flow to the forearm in order to induce ischemic pain. Doing this causes a gradually increasing deep, diffuse, dull aching muscle pain. The tourniquet cuff was placed on the subject’s arm above the elbow. The arm was supported and elevated vertically for 30 seconds to promote venous drainage and the cuff was then rapidly inflated to 200 mmHg. The arm was then lowered to rest on a pillow on the arm of a chair and the subject completed 20 handgrip exercises with 30% maximum force for two second durations each, with an inter-squeeze interval of two seconds. When the subjects initially felt a sensation of pain they were instructed to say “pain” and to say “stop” when they were no longer able to tolerate the pain. Immediately after the subjects rated the aspects of pain intensity from 0 to 100 being the most intense sensation imaginable. They also rated unpleasantness from 0 to 100 being the worst pain imaginable. The maximum time limit for this test was 20 minutes to prevent tissue damage and the time to pain threshold and tolerance were recorded.

To perform the Hand cold pressor task, a container was filled with ice and water, which was maintained at 4°C, and a water circulator was placed inside the container to prevent the water from warming near the subject’s hand. The subjects placed their hand in the container to a marked line on their wrist and remined still. The maximum time limit for this test was 5 minutes to prevent tissue damage. The time to pain threshold was recorded and intensity and unpleasantness was accessed.

To perform the Thermal heat pain task, a 1cm diameter contact thromde was created. The threshold and tolerance were determined by increasing the diode temperature in .5°C increments every 5 seconds, stating at 41.5°C until it reached 53°C or until the subjects reach tolerance. The average of the three series were calculated to determine the threshold and tolerance.

African American women demonstrated significantly lower pain tolerance to hand cold pressor pains and a marginally lower tolerance to ischemic pain when compared to non-Hispanic Whites. Based on the ethnic difference in oxytocin concentration, there was no ethnic difference in tolerance to thermal heat pain. African American women had significantly lower baseline plasma OT concentrations that non- Hispanic White women and the levels of OT were also lower in African American compared to Whites. From the relationships of OT to other biological measures, in African Americans higher OT concentrations were significantly correlated with lower systolic blood pressure.

Oxytocin is a powerful hormone that cats as a neurotransmitter in the brain. It is known as the “love drug” as it is associated with feeling good. It is manufactured at many central and peripheral sites, but the paraventricular nucleus of the hypothalamus has been identified as its primary source. It is best known for its milk let down function in lactation, and uterine contraction during parturition. The relationship between oxytocin concentrations and lesser pain sensitivity has been supported. Oxytocin and pain tolerance have an inversely proportional relationship.

This experiment was designed to test the relationship between endogenous OT concentrations and sensitivity to experimental pain in humans. Pre-menopausal women with. Higher circulating OT levels during resting baseline demonstrated greater tolerance ischemic pain and cold pressor pain. There is indeed a link between lower OT levels and reports of greater clinical pain. African American not only differed in OT levels but also in pain- response profile. African Americans demonstrated lower tolerance levels to ischemic, cold pressor and thermal heat pain than their white counterparts. These finding support the theory that differences in OT may be one of various underlying biological mechanisms contributing to the observed ethnic differences in types of clinical and experimental pain. Greater pain sensitivity in African Americans were present regardless of whether pain testing was done after stress or after rest. It can be concluded that reduced oxytocinergic function may be one of many factors underlying pain experiment by African Americans compared to Whites. Lower OT levels and decreased pain tolerance in African American women may reflect the long -term effects of physiological responses to physiosocial stress.

Reference:

Grewen, KM, Light ,KC, Mechlin, B, Girdler, SS. Ethnicity is associated with alterations in oxytocin relationships to pain sensitivity in women. Ethn Health. [Internet]. 2008[October 17, 2020];13(3):219-241. doi: 10.1080/13557850701837310

Carmen Major

Senior Prospectus

February 7, 2021

Pregnancy Related Mortality in The United States, 2011-2013

Over the past years, there has been an increased mortality rate for African American women. The first factor is discrimination, especially based on race. African American women suffering from obesity do not get health care services. The economic status and the general poverty amongst these women have also contributed to their high mortality rate. An observational study method was used to collect the data, and there was evidence that there was a high mortality rate for the women.

African American mortality rate is high in the USA because of issues about the system and inequalities that these people are facing. A critical assessment about the societal and issues that stem from health care and their role in the increased mortality rate of African American women and the connection of their death to obesity despite many other factors that contribute to this mortality rate. African American women must undergo prenatal care since it is vital in promoting pregnancy, which is healthy, reducing maternal mortality. There is critical prenatal care which these women were not getting access to, and this was making the women suffer; the critical elements included education to the parent during pregnancy, screening of both mental health and anything that may pose a challenge to them as well as being sure that the general health aspects have been addressed.

African Americans are likely to die from obesity. This as well can be linked to poor maternal health. This is because if the disorder is treated, it cannot kill these women. The main focus of high mortality rate is the issues of health complications. This is because of the behavior and the facts which health care practitioners have towards African American women. However, other issues have been seen as the cause of high mortality rates, and they include smoking and drug abuse, which in most instances lead to high mortality rates.

As part of the methodology, observational studies were used to achieve the results. The one thing that has been dominant in the studies was racial discrimination. The other test that was done included looking at the ten women whom after they were assessed it was seen that they were not attending clinics and they were not being given treatment.

After the author used population-based to check data from pregnancy and mortality surveillance system to calculate any pregnancy-related mortality in all the years through using the age group and race-ethnicity group, it was found that the most cause for complications and maternal mortality was smoking and drug abuse. The abuse was very high amongst African American women, especially those at that age of childbearing. This had many complications during childbirth, such as children born underweight, the pattern and delivery were not healthy. All the factors above had some influences on the mortality rate.

The article has analyzed many causes of death in African American women, linked with obesity and many other factors. The article analyzed the trend for education amongst these women, and this lack of knowledge on maternal issues and how to deal with lifestyle diseases is a link to maternal mortality.

References

Creanga, A. A., Syverson, C., Seed, K., & Callaghan, W. M. (2017). Pregnancy-related mortality in the United States, 2011–2013. Obstetrics and gynecology, 130(2), 366.

Biol 491 Senior Seminar

Carmen Major

September 17, 2020

Prostate Cancer in African American Men Is Associated with Downregulation of Zinc Transporters

In the United States, African American men have a 35% higher incidence rate and a 223% higher mortality rate due to prostate cancer when compared to other racial groups. This is due to downregulation of zinc transporters and the loss of the ability to retain normal intracellular levels of zinc. Africa is a mineral-rich continent and the levels of zinc are very high. It was hypothesized that Africans may have genetically decreased the quantity of zinc absorption capacity so they would not absorb abnormal amounts of zinc. Zinc is an essential nutrient to all organisms because it is a required catalytic cofactor of zinc dependent enzymes. Absorbing abnormal amounts of zinc could lead to death in certain parts of the brain and other disorders.

As a result, the lower the levels of zinc, the higher the rate of prostate cancer. To test this hypothesis, 58 prostate cancer tissues were evaluated from two major racial groups (30 from Caucasians and 28 from African Americans) to see if they were able to express two major human zinc transporters, hZIP1 and hZIP2, that are responsible for accumulation of zinc in the prostate glands. Reverse transcriptase in situ polymerase chain reaction was used as well as semiquantitative reverse- transcriptase polymerase chain reaction (SQ-RT-PCR). SQ-RT-PCR was used to show the standard curves of in vitro transcribed mRNAs of hZIP2. By using semiquantitative reverse- transcriptase polymerase chain reactions, it showed that zinc ions are produced and discharged from the epithelial cells into the acinic lumen and the intercellular canaliculi.

Prostate secretory epithelial cells are able to accumulate high intracellular levels of zinc, which can induce mitochondrial apoptogenesis. The prostate contains high amounts of free zinc ions that are excreted into seminal fluid. The expression of zinc transporters also appears to be regulated by prolactin, a peptide hormone produced by the pituitary gland that causes breast to grow and develop milk, and testosterone, a hormone produced by the human body that stimulates spermatozoan production in men.

The specimens from the Caucasian men showed a significantly higher degree of expression of the two zinc transporters than the African American men. All 30 of the specimens from the Caucasian men biopsies displayed a modest degree of expression. From the African American men 26 out of the 28 men displayed a very low expression of both of the zinc transporters. In one of the other two, there was a high expression of hZIP1 and a low expression of hZIP2 and vice versa. The hypothesis stands that there are differences in the zinc transport in different racial groups.

Carmen Major

February 16, 2021

Senior Seminar

How Implicit Bias Contributes to Racial Disparities in Maternal Morbidity and Mortality in the United States

In the United States, approximately 700 women die each year due to complications during pregnancy and delivery. The pregnancy related mortality ratio varies significantly by race. There has been a 50% increase in the US maternal mortality rate over the same time period. From 2011 to 2015, White women experience 13 deaths per 100,000 births, while black women experience 42.8 deaths per 100,000 births. The goal of this article is to review implicit bias and its impact that it can have on healthcare and health disparities.

Every three and five pregnancy related deaths could have been prevented. Implicit bias is defined as thoughts and feelings that exist outside of conscious awareness and subsequently can affect human understanding, actions, and decisions unknowingly. Implicit bias developed early in life from exposure to repeated reinforcement of stereotypes. Implicit bias has permeated into the U.S. healthcare system and is a contributing factor to the high negative maternal health outcome experienced by African American women. The Implicit Association Test is a tool that can be used to assess implicit bias. It is available online and is computerized.

Implicit bias is directly correlated with lower quality of healthcare for patients. Implicit bias can also affect how providers communicate with their patients. It is said that the reason of implicit bias in the healthcare system could be due to an overload or high stress. Being in the medical profession, one strives for equal treatment of all patients, but racial disparities are prevalent in the healthcare system. Racial disparities are prevalent because implicit bias affects the providers perception, which create inequalities in access treatment decisions, and health outcomes. Some providers hold fast believes about biological differences between African American and Caucasian individuals that increase the bias. Some believe that African American people have less sensitive nerve endings, thicker skin, and stronger bones. Due to these beliefs, providers tend to rate African American patient pain lower which results in less appropriate treatment recommendations.

Approximately one and five black and Hispanic women experience miss treatment from hospital-based care providers because of their race, ethnicity, cultural background, and or language. This has led many women to avoid the hospital if possible and utilize midwives in dollars for at home or birth center services.

It is important to improve communication and the need for healthcare providers to be aware of their own implicit biases. To resolve racial disparities in maternal health many things can be done. Practicing cultural humility eases implicit bias, promotes empathy, and edifies healthcare providers in acknowledging and respecting patient individuality. Cultural humility stresses that providers should make a connection with their patients other than assuming. Awareness of implicit bias should be incorporated in medical and nursing schools as well as residency training programs to train the next generation of healthcare providers to treat patients with respect and to provide high-quality care. Hospital systems should also implement mandatory Maternal Mortality and Morbidity reviews which can help identify areas of care that need improvement.

Reference

Saluja, B., Bryant Z. How Implicit Bias Contributes to Racial Disparities in Maternal Morbidity and Mortality in the United States. Journal of Women’s Health [Internet]. 2021; 30(2):270 -273 doi:10.1089/jwh.2020.8874