Experiment 3 Report Make Predictions about chitobiase activity of mutant chb gene and measure chitobiase activity using

an in vitro assay

Soojung Kim 4/15/14

BMB 445W Experiment 3 Part A Introduction In its capacity as a repository of information, a DNA molecules most important property is its nucleotide sequence. The development of new techniques in the 1970s, made possible the sequencing of larger DNA molecules with ease unimagined just a few years before. In a DNA cycling sequencing reaction (Sanger method), each dideoxynucleotide can be linked to a fluorescent molecule that gives all the fragments terminating in that nucleotide a particular color. All four labeled ddNTPs are added to a single tube. The resulting colored DNA fragments are then separated by size in a single electrophoretic gel contained in a capillary tube. All fragments of a given length migrate through the capillary gel in a single peak, and the color associated with each peak is detected using a laser beam. The DNA sequence is read by determining the sequence of colors in the peaks as they pass the detector. This information is fed directly to a computer, which determines the sequence. Today, entire genomes of more than a thousand organisms have now been sequenced in this way, and many very large DNA-sequencing projects have been completed or are in progress. Sequencing of DNA molecules containing thousands of nucleotides can be completed in a few hours. The goal of Experiment 3 is to use DNA sequencing to analyze the unique chb gene for which its SDS category has not been determined and is distinguished from the Category 1 mutant. Experiments 1 and 2 has allowed isolation and cloning of this particular chb gene, and in Experiment 3, alignments are performed to compare the chb gene sequence with sequences from other related genes. Cloning and sequencing chb genes into pBMB and pBMB1 has allowed scientists to measure chitobiase activity and discover that Category 1 outbreaks express a chitobiase enzyme with an altered substrate specificity. Similarly, we want to use the sequence data to determine the molecular characteristics for this unique chb gene and how it categorizes within the SDS outbreaks. For the sequencing of the chb gene, amino acid sequences for 1) V.harveyi chitobiase protein 2) Homo sapiens beta-hexoaminidase subunit alpha preproprotein 3) Serratia marcescens chitobiase protein are obtained from the NCBI protein sequence database (http://www.ncbi.nlm.nih.gov/protein). The V.harveyi chitobiase amino acid sequence is aligned with these two other glycosyl hydrolase 20 family members. By comparing across taxa, information on the conservation of certain residues can be obtained. Also, the Serratia marcescnes chitobiase protein sequence is used because it has already been crystallized, which allows easy access to the structure of the protein for the prediction of importance of certain amino acid residues. The alignment uses a CLUSTAL omega alignment method, a new method which can align virtually any number of protein sequences quickly and that delivers accurate alignments. The alignement percent identity matrix shows that V.harveyi chitobiase protein and Serratia marcescens chitobiase protein are most similar (55.31%) while V.harveyi chitobiase and the human HexA protein are the least similar (22.37%). In the alignment it can be seen that some regions are relatively well conserved, showing little or no change in amino acid identity or property. Amino acids such as Arg349, Asp 539, Glu540, Tyr669, and Trp616 have important roles in the structure / catalysis of S.marcescens chitobiase protein, and are most likely to be conserved. Other regions in which neither amino acid identity nor property is conserved indicate that amino acids in these regions are nonessential and can be varied without affecting the function of the protein.

Once the different proteins are compared, alignment of the nucleotides sequences from pBMB2 and wild type V.harveyi is performed. This allows the determination of any mismatches and mutations that are found in the pBMB2 unique chb gene. Any nucleotide change or mismatch (mutation) will affect the codon and subsequently the amino acid / protein structure. Nucleotide mutations within the coding sequences causing substantial amino acid change will result in alternation of the proteins catalytic activity. All of the nucleotide mutations found in the alignment between pBMB2 and wild type V.harveyi are analyzed as to whether it is located in the coding or noncoding region, what amino acid changed, how the mutation is categorized, and lastly which amino acid it aligns with in Serratia chitobiase. Table 1 shows the list of mutations and the possible changes it might have on the chitobiase protein. Referring to the Serratia marcescnes chitobiase protein crystallized structure (Tews et al., 1996) the amino acid alignment from the mutation can be compared to the Serratia chitobiase and the effect the mutation will have on the protein enzyme activity can be predicted as well. Amino Acid that aligns in Serratia chitobiase

Nucleotide Mutation g.493 C>G

Coding or Noncoding

g.2721 C>G

Categorization of Mutation Missense Coding A16G G20 (Nonconserved) Predicted effect of mutation: The protein structure and catabolic activity is likely to be slightly altered and reduced. Missense Coding Q759E E760 (Nonconserved) Predicted effect of mutation: The protein structure and catabolic activity is likely to be altered and reduced. Noncoding N/A N/A N/A Predicted effect of mutation: This mutation located in the noncoding region of the gene is not likely to affect the structure or catalytic activity of the protein. Coding D536_E537del Deletion D539_E540 Predicted effect of mutation: Mutation that deletes the essential functioning amino acid of Glu540 will cause decrease in activity.

Amino Acid Changed?

g.3149_3150 TT>AA

g.2053_2058 Del

Table 1: pBMB2 chb gene Mutation Analysis: the mismatches of alignments are analyzed to determine the amino acid change, category of mutation, and amino acid alignment with Serratia chitobiase.

The g.493 C>G and g.2721 C>G mutations both cause missense nonconserved amino acid changed within the coding region. g.493 C>G causes the amino acid at position 16 to change from Alanine to Glycine. This change is likely to alter the protein sequence rather less substantially because both Glycine and Alanine are amino acids with small molecular weights and nonpolar side chains. The physiochemical change from Ala to Gly will indeed cause a change in the protein but the extent of it will not be drastic. The g.2721 C>G mutation causes the amino acid at 759 to change from Glutamine to Glutamate. Gln and Glu are extremely similar in molecular weight (both approximately MW:128) and the similarity in size and functional groups is likely to leave the protein structure itself relatively unchanged. Still, Glu has an electrically

charged side chain, while Gln has a polar, less acidic side chain and this change in amino acid property will likely cause the catalytic activity of the protein to be reduced. Based on the Tews et al. paper, the Asp539 and Glu540 have important interactions with the chiboaise active site. Glu540 is the carboxylate residue acting as the catalytic acid in chitobiase and binds to the glycosidic linkage. Therefore, Glu540 is invariant among family 20 enzymes, and in chitobiase, there is no second protein carboxyl group at an appropriate distance for either a retaining or for inverting mechanism. Based on the crystal structure, the research group proposes that chitobiase uses an acid-base reaction mechanism with Glu540 as the catalytic acid and with the N-acetyl group of the substrate replacing the enzymatic nucleophilic base found in other glycosyl hydrolases. Therefore, the g.2053_2058 del mutation found from the alignment will severely alter the enzyme activity in the pBMB2 and differentiate it from the category 1 SDS chb genes. In addition to sequencing, chitobiase assay is to be performed. The pBMB2 transformation cultures harvested will be treated with Sarkosyl to extract the chitobiase enzyme from the bacteria, allowing the SCE to be used as the chitobiase enzyme source. The assays are conducted in 10mM Tris-HCl, pH7.3 and 666uM PNAG at 25C. Enzyme is added at zero time to each reaction mixture and incubated. At different time intervals, the absorption of the assay reaction is determined by a spectrophotometer. This assay uses the basics of the rate of pnitrophenol formation from p-nitrophenyl-acetamido-2-deoxy--D-glucopyranoside (PNAG). PNAG is a used as an effective substrate because the product formed from PNAG can be observed by color and measured with the spectrophotometer. The chitobiase enzyme catalyzes PNAG into N-acetyl glucosamine and p-nitrophenol. While p-nitrophenol is colorless at acidic pH values, increased pH of the reaction medium will allow yellow p-nitrophenolate to form of which the absorbance can be measured and monitoredat 400nm. In the protocol specific for Experiment 3, sarcosyl cell extracts for wild type chb gene (pBMB), cloned pBMB2, and pUC19 cells are prepared and used as sources of the chitobiase enzyme. Each extract is added to the Master mix that consists of the 10mM Tris-Cl, pH7.3 and 12mM PNAG. The change in absorption at determined time intervals are measured using the Genesys Specs. The data obtained from the assay can be used to determine the enzyme activity and specific activity of the chitobiase enzyme using Beer-Lambert Law A= abc.

References 1. Tews, Ivo, Anastassis Perrakis, Amos Oppenheim, Zbigniew Dauter, Keith S. Wilson, and Constantin E. Vorgias. "Bacterial Chitobiase Structure Provides Insight into Catalytic Mechanism and the Basis of TaySachs Disease." Nature Structural Biology 3.7 (1996): 638-48. Web. 2. Soto-Gil, Rafael W., and Judith W. Zyskind. "N,N'-Diacetylchitobiase of Vibrio Harveyi." The Journal of Biological Chemistry 264.25 (1989): 14778-4783. Web. 11 Apr. 2014.

BMB 445W Experiment 3 Part B Soojung Kim and Lismari Reyes-Munoz Reagents List 10.4 mL of 10mM Tris-Cl, pH 7.3 610 ul of 12mM PNAG (PNAG dissolved in Tris-Cl, pH 7.3) 50 ul of 1% Sarcosyl in Tris, pH 7.3 1.5 ml cuvettes (5) 15 ml centrifuge tube (1) Parafilm Genesys Specs set up at 400 nm 1-2 10 ml plastic pipette for Master Mix preparation 1. Take 4 of the 1.5ml cuvettes and label each cuvette as: a) pBMB b) pBMB2 c) pUC19 d) Blank 2. In a 15ml Centrifuge Tube, prepare the Master Mix containing the 10mM Tris-Cl, pH 7.3 buffer and the 12mM PNAG substrate following the recipe below: a) Add 610 ul of 12mM PNAG to 10.4 mL of 10mM Tris-Cl, pH 7.3 to yield 11ml of Master Mix (666uM PNAG in 10mM Tris-Cl, pH 7.3). Make sure to mix the components well by pipetting up and down or inverting the tube a few times. 3. Pipette 1ml of the Master Mix to each cuvette. Parafilm the remaining Master mixture in case you need it later. 4. The Blank solution should not have any substrate converting into product. In order to make sure there is no enzyme added but still is as close to the experimental samples, 30ul of 1% Sarcosyl in Tris, pH7.3 (solution buffer used to prepare the Sarcosyl Cell Extracts) will be added to the blank. Mix the contents well by pipetting up and down and set aside. 5. Prepare the Genesys Specs for measurement. It will take a few seconds for the machine to be ready to take sample. Set the wavelength at 400nm for the measurement and make sure the machine is ready before preparing the samples. The Blank solution should be placed in the first sample holder of the Genesys Specs. 6. Add the appropriate enzyme to the cuvette as listed. Make sure that the enzymes are added to the all cuvettes with little time interval in between so that experimental data is not affected by time. Make sure to mix each solution well by pipetting up and down. a) pBMB2: Add 30 ul of pBMB2 Sarcosyl Cell Extract b) pBMB: Add 30 ul of pBMB Sarcosyl Cell Extract c) pUC19: Add 30 ul of pUC19 Sarcosyl Cell Extract 7. Immediately after the enzymes are added, the cuvettes are inserted into the Genesys Specs to measure the absorbance. Make sure to record the absorbance values in your notebook. The autoprint option in the Genesys Specs system can be selected to get a print-out of the values as well. 8. Repeat step 7 at 5, 10, 15, and 20 minute time intervals measuring the appropriate cuvette absorbance along with the two controls.

9. Duplicate the assay: Repeat steps 4-8 once more and record the measurement that can be compared to the first trial. (The Master Mix is enough to carry out the measurement three times, in case the data are not consistent) 10. For each time point measurement, take the average of the absorbance values taken from each trial measurement. A sample table for record-keeping is shown below. Absorbance (400nm) Time (min) Trail 1 Trail 2 Trial 3 Average 0 min 5 min 10 min 15 min 20 min 11. Using these average values, produce an Absorbance vs. Time(min) plot on Excel. Determine the trend-line and equation for the graph. 12. A graph for the positive control pBMB must also be constructed for comparison with the experimental value. 13. Analysis of Graph: a) From the equation of the graph, determine the slope of the Absorbance vs. Time graphs. This slope is in units of Abs/ minutes (or Abs/ sec) b) Using the Beer-Lambert Law A= abc, you can find the concentration. A is absorbance, a is the molar absorbtivity (L mol-1 cm-1), b is the path length of the sample (cm), and c is the concentration of the compound in solution (mol L-1) Since here you start with the slope of the graph (Absorbance per time) you will be able to find concentration per time (moles of product formed per minute or second) c) The concentration per time is the velocity of the enzymatic reaction. One unit of chitobiase activity is defined as the amount of enzyme that catalyzes the formation of 1umol of p-nitrophenol in 1 min in the assay. Specific activity is expressed as units per milligram of protein. d) Multiply both sides of the equation by the cuvette volume and 106 umoles/mole (since the molar absorbtivity is expressed in moles and the enzyme activity is expressed in umols). This value represents the total enzyme activity in the cuvette. e) You can calculate the specific activity by dividing the total activity by the total amount of protein enzyme added to the cuvette.

Experiment 3 Part C Results In earlier experimental procedures, the unique chb gene from the 092308. C6 SDS isolate was cloned, sequenced, and analyzed for mutations that differentiates it from the wild type V. harveyi chb gene. Because Shell Disease Syndrom appears to center around chitin degradation, it was suggested that the categorical difference of this particular SDS originates from alterations in protein structure and function. The nucleotide and amino acid alignment allowed predictions be made on the effect the mutations have on the chitobiase protein. The in vitro chitobiase assays provides a means to verify the prediction; the nucleotide mutations and amino acid changes will cause the chitobiase enzyme to have a significantly decreased activity. Before carrying out the chitobiase assay of the pBMB, pUC19, and pBMB2 SCE samples, a BCA protein assay was carried out in order to determine the total protein concentrations of all SCEs. The BCA assay exhibits protein concentration by a color change of the sample solution from green to purple in proportion to the total protein. By measuring BSA standards with the SCE samples, a BSA Standard Curve was constructed (Supplemental Graph 1*). The trendline equation of this graph y= 0.3091x -0.1975 was used to calculate the total concentration of each SCE from their absorbance values. Table 1 shows the original concentrations of SCEs calculated using the standard curve equation, and the diluted concentrations of SCEs to normalize for the lowest concentration of protein. SCE Sample pBMB2 pBMB pUC19 Original Total Protein Concentration 3.96 mg/ml 4.94 mg/ml 2.58 mg/ml Concentration After Dilution 2.58 mg/ml (65 ul of SCE added to 35 ul of 1% Sarcosyl in Tris for dilution) 2.58 mg/ml (52 ul of SCE added to 48 ul of 1% Sarcosyl in Tris for dilution) 2.58 mg/ml

!able 2: Original Total Protein Concentrations and Diluted Concentrations of Sarcosyl Cell Extracts

The initial chitobiase assay protocol was designed to measure chitobiase activity using 30ul of Sarcosyl Cell Extracts in 970ul of MasterMix (10mM Tris-CL, pH7.3 and 666uM PNAG) for a total volume of 1ml at 25C. The first trial of the assay revealed that 30ul of pBMB SCE (positive control) contained a relatively high concentration of enzyme, causing the absorbance of the assay sample to spike up rapidly and reach saturation before comparable measurements can be made in other SCE samples. Therefore, the protocol was adjusted so that 10ul of Sarcosyl Cell Extracts are added to 990ul of Master Mix and absorbance measurements (400 nm) made at t = 0, 3, 5, 8, 10, 15 minutes instead of the previous longer time intervals of t = 0, 5, 10, 15, 20 minutes. The positive control still showed a relatively quick increase in absorbance, but time to reach the absorbance plateau was extended enough to observe changes in absorbances of other SCE samples. Samples 1, 2, 3 were used for the pBMB2 SCE for reproducibility of data. Table 2 shows the absorbance values for each SCE at indicated time intervals. The pUC19 SCE (negative control) does not contain the chb gene and is, therefore, not expected to show any chitobiase enzyme activity. The pBMB SCE on the other hand, contains the wild type chb gene and should have the most active chitobiase enzymes with obvious catalytic activity. This is supported by the

data, showing that the absorbance values of pUC19 SCE show very little change, in fact a decrease in absorbance, while that of pBMB SCE is quickly increased in time. The SCE of pBMB2 samples displayed no change in absorbance and thus no chitobiase enzyme activity. This result was consistent in all replicate measurements. SCE pUC19 pBMB pBMB2 (1) pBMB2 (2) pBMB2 (3) t=0 0.020 0.414 0.014 0.009 0.006 t=3 0.017 1.215 0.015 0.008 0.005 Absorbance (400 nm) t=5 t=8 0.017 1.642 0.015 0.008 0.005 0.016 1.962 0.015 0.008 0.005 t = 10 0.016 2.030 0.015 0.008 0.005 t = 15 0.016 2.062 0.015 0.008 0.004

Table 3: Absorbance Values of Sarcosyl Cell Extracts at Different Time Points

The absorbance values of each SCE can also be plotted on a graph of absorbance over time as shown in Figure 1 below. The graph of pBMB absorbance changes shows a logarithmic curve that increases until it reaches a plateau approximately around 2.0; at this point the absorbance does not further increase because the active sites of enzymes are saturated. Graphs for all other SCE show a horizontal linear trend with a slope of 0 since no chance in absorbance is observed.
!

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Figure 1: Absorbance (400nm) vs. Time (min.) of Sarcosyl Cell Extracts The graph for pBMB, the positive control, shows a logarithmic trend with changes of absorbance. The remaining SCE samples show no change or increase in absorbance, suggesting absence or inhibition of enzyme activity.

Using the Beers Law equation of A=!bc, where ! = molar absorptivity coefficient (L mol-1 cm1 ), b = path length of the sample (cm), and c = the concentration of the compound in solution (mol L-1), the absorbance values can be converted to unit and specific activity values. Table 3 provides units and specific activity for each SCE sample at two different time intervals. Because the negative control pUC19 and the experimental samples pBMB2 (1), (2), (3) show no change in absorbance, the unit and specific activity values for these SCEs remain at 0. The units and specific activity values for pBMB at the two different time points are slightly different. This is due to the fact that the pBMB absorbance graph takes a logarithmic curve with instantaneous rates of change differing at each point of the graph. Since the rate of change in absorbance is larger for earlier time periods and gradually decreased over time, the units and specific activity values are smaller for the later time period t=3 to t=8. Example calculation for converting absorbance values to units and specific activity is provided below as well. Time period (1): t=3 to t=5 SCE Sample pUC19 pBMB pBMB2 (1) pBMB2 (2) pBMB2 (3) Unit 0 1.67x10 umoles/min 0 0 0
-2

Time period (2): t=3 to t=8 Unit 0 1.17x10 umoles/min 0 0 0

-2

Specific Activity 0 0.646 Units/mg 0 0 0

Specific Activity 0 0.452 Units/mg 0 0 0

Table 4: Units and Specific Activity values of Sarcosyl Cell Extracts at Two Different Time Periods

Sample Calculation: pBMB at time period t=3 to t=5 Concentration of Sarcosyl cell extract stock: 2.58 mg/ml Amount of SCE added to each individual sample: 10 ul Absorbance measured at t=3: 1.215 Absorbance measured at t=5: 1.642 ("Absorbance = 0.427) ! = 12,800 M-1cm-1 ! !!!"# !! ! ! !!!!"!!"!! ! ! !!! !"!!""! ! !"!! ! !"#
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Discussion Through nucleotide and amino acid alignment, it was predicted that the missense mutations that resulted in amino acid changes and deletion of the essential functioning Glu540 will significantly decrease the chitobiase enzyme activity of the SDS isolate. This hypothesis was supported by the chitobiase assay results, as the pBMB2 SCE samples (1), (2), and (3) showed zero unit and specific activity. The mutations in amino acid sequences seemed to have completely destroyed the enzyme catalytic activity. This result, then, suggests that this type of SDS is different from the classical SDS, which has mainly associated its disease progression with chitin degradation. Rather, chitoprotein degradation seems to be of less importance in this more severe and high-mortality form of SDS. Previous studies of SDS have suggested that lesions act as microniches occupied by various microbial groups whose interactive effect result in lesion progression and that SDS is a collective effect of microbial communities (Vogan et al., 2008). Some microbes are active enzyme producers, while others act as scavengers and predators. It is likely that the dominant microbial species affecting this kind of SDS is a non-chitin feeder that contributes to the disease progression through mechanisms other than chitin degradation activity. Smolowitz (2005) acknowledged the new form of shell disease as Epizootic Shell Disease (ESD), which, unlike other known forms of shell disease, is characterized by severe, deep erosions of the cephalothorax and abdomen that spread irregularly over the dorsal carapace of the animal. Necropsy, tissue processing, and microscopic evaluation of ESD infected crustaceans revealed that the invading bacteria of ESD are not primarily chitin feeders, but instead were attracted to the proteins and lipids making up the matrix. The etiology of ESD is not conclusive, but Smolowitz suggests that the microbes present in ESD shell lesions are likely to contribute to the biofouling of the carapace. This is a clear distinction from classic SDS, as chitin degradation appears to occur minimally in lesions, while mainly lipolytic and proteolytic activities of bacteria degrade the carapace to produce lesions. Conclusions about whether this specific SDS isolate is a definite category of ESD cannot be made; the data table describing the SDS isolate genomic samples (Lab Manual pg.16) describes lesions being average in size and number while ESD usually shows lesions larger and more extensive. However, knowing that there is a category of SDS that is affected less by chitinoclastic bacteria and more by various biofouling, opportunistic microbes, it can be suggested that this unique form of SDS can be contributed to the activity of biofouling, lipolytic, proteolytic bacteria that cause erosions of not only the chitin-containing exoskeleton but lipid and protein-containing matrix and ineternal layers, leading to a more serious damage to the organism. The erosions are likely to create a microniche and rough surfaces that allow for colonization of various epibionts and microbes, causing the disease to progress further. As there are various microbial communities that affect SDS, the cause of the disease itself cannot be contributed to one bacterial colony or defining factor. There are also other factors to consider when evaluating the different categories of SDS. Stressful environment can compromise the immune system of the organism, it might change the characteristics of the pathogen or dynamics of the bacterial community. Increased temperature of the marine environment can promote the growth of bacteria. The frequency of molting by organisms can influence the colonization of microbes on their surfaces. As listed above, there are multiple components that lead to this complex disease of SDS, including both infectious and non-infectious factors. The mechanism of SDS progression is not fully understood and its complexity makes the disease difficult to treat. However, certain measures can be taken to reduce the possibility of SDS infection. Basic care of

crustaceans such as in nutrition, temperature, water quality, pollutant elimination, etc. should be carefully monitored. Since the severity of the disease is dependent on the presence of a diverse microbial community, UV treatment /purification of water at the aquafarm can irradiate bacteria and viruses. This measure does not directly eliminate the agents causing lesions and erosions, but could potentially prevent the progression of SDS that is further propelled by the microbial communities colonizing the surfaces of crustaceans. Still, such conventional approaches will have limited success in the prevention or cure of aquatic disease. Methods that are more promising are the use of vaccines and probiotics. Immunostimulants in combination with vaccines can enhance the defense mechanism of crustaceans. The use of probiotics can stimulate the crustaceans to abundantly establish symbiotic host-microbe relationships, so that those pre-existing symbioses prevent colonization and proliferation of pathogenic microbes. Manipulating microbial colonies in aquatic species is trickier than for mammals, since hosts and microorganisms share the environment from the start. However, if a temporary change in the composition of microbial community can be established, the symbiotic relationships can be further encouraged by repeated contact with the symbiont. As mentioned in the Kremer et al. (2013) paper, early interactions between a bacterial symbiont and its host are crucial for establishing a long-term relationship. Therefore, if the acaufarm microbial environment can be controlled with probiotics to prime the crustaceans for symbiotic microbes, the non-specific growth biofouling microbial species could possibly be prevented. Some methods could also utilize the practical aspects of regulation of gene expression. As it has been previously demonstrated in Larsen et al. that chitinolytic activity of microbial pathogens is further activated by the presence of chitobiose or GlcNAc. Then, in order to prevent further activation of chb genes and chitoprotein, removal of such substrates could be helpful in SDS control. A specifically engineered enzyme that degrades or a chemical designed to bind and cause some sort of conformational change in the GlcNAc substrate could be suggested. In this chitobiase assay, the exact amount of pure chitobiase could not be experimentally determined since the SCEs contain other various proteins. The SCE concentrations are therefore normalized for the concentration of pUC19 SCE, as it represents the total protein except for the chitobiase enzyme. In doing so, the assumption that all SCEs contain the same kind and same amount of proteins is made. This may not be the most accurate representation of chitobiase concentration, as even though the concentrations are normalized, the measurement is against a body of different proteins, among which chitobiase must only comprise an insignificant portion. Plasmids pBMB and pBMB2 are both constructed from pUC19 and therefore all three plasmids contain the same structural components. However, pBMB and pBMB2 have the inserts disturbing the MCS of the vector; this could induce nonspecific protein products that are not normally expressed in the pUC19 alone or result in the complete opposite of eliminating certain gene expression products. Thus, normalization of concentration does not ensure that measured enzyme activity is solely from chitobiase protein. There needs to be a measure to selectively assay chitobiase protein.

References 1. Smolowitz, R., Chistoserdov, A.Y., and Hsu, A. (2005) A description of the pathology of epizootic shell disease in the American lobster, Homarus americanus, H. Milne Edwards 1837. J Shell#sh Res 24: 749756. 2. Vogan, Claire L., Adam Powell, and Andrew F. Rowley. "Shell Disease in Crustaceans Just Chitin Recycling Gone Wrong?" Environmental Microbiology 10.4 (2008): 826-35. Print. 3. Larsen, M. H., J. J. Leisner, and H. Ingmer. "The Chitinolytic Activity of Listeria Monocytogenes EGD Is Regulated by Carbohydrates but Also by the Virulence Regulator PrfA." Applied and Environmental Microbiology 76.19 (2010): 6470-476. Print. 4. Wernegreen, Jennifer J. "First Impressions in a Glowing Host-Microbe Partnership." Cell Host & Microbe 14.2 (2013): 121-23. Print.

Enzyme Assay BSA Standard Curve

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Supplemental Graph 1: BSA Standard Curve The absorbance measurements of BSA standard samples were used to construct a BSA Standard Curve. The trendline equation is used to calculate the total protein concentration of each SCE. The absorbance measurement of the 0.5mg/ml BSA sample was omitted from the standard curve for a best-fitting trend line.