Experiment 1 Report

Develop a rapid screening kit that can detect category 1 SDS outbreaks and use the kit to try and categorize unknown samples of SDS

Soojung Kim 2/21/14

BMB445W: Experiment 1 Write Up Part A: Internal Report To: Supervisor From: Soojung Kim Subject: Category 1 SDS Analysis and Screening Kit Report Date: February 20, 2014 The purpose of this report is to present my analysis of the Vibrio. harveyi chb gene and the progress made in the development of the Shell Disease Syndrome Screening Kit. The goal of the screening kit is to detect Category 1 SDS isolates, and once marketed, the kit will be useful for other aquaculture companies and marine scientists interested in tracking and classifying SDS outbreaks. Analyses of SDS genomic samples 092308.C6 and 070505.A1 provided preliminary data for the screening kit, and has projected its potential in the market. Section 1 Atlantis Aquafarm, Inc., one of our companys biggest clients, has been experiencing a severe case of Shell Disease Syndrome outbreaks and requested our research team to develop a screening kit for Category 1 SDS. Shell disease syndrome is a condition that affects a wide range of crustaceans, resulting in the bacterial degradation of the exoskeleton causing lesions and even death. It is known from studies that the disease syndrome results from activities of chitin-destroying microorganisms. The chb gene from microbe Vibrio Harveyi encodes protein N,N-diacetylchitobiase that degrades chitin, and it is this very gene that is analyzed and assayed for in order to categorize the SDS outbreak. Through previous research experiments, the chb gene has been cloned in E.coli and isolated (Jannatipour et al. (1987) Journal of Bacteriology, 169: 3785-3791). This nucleotide sequence of Vibrio harveyi chb gene wildtype is indexed and can be found on the NCBI Gquery nucleotide database (GI: 155154). Groups of scientists also developed plasmids pBMB (wild type copy of the chb gene plus flanking regulatory sequences) and pBMB1 (Category 1 chb gene cloned into the pUC19). The important objective of our research was to analyze the wild type chb chitobiase sequence in comparison with the Category 1 Vibrio harveyi chb sequence. Using the BLAST (Basic Local Alignment Search Tool) program, the wild-type V.harveyi chb gene sequence from the Gquery nucleotide database and the Category 1 chb gene sequence from pBMB1were aligned and compared. The alignment allowed determination of the mutations in the Category 1 chb gene that differentiated it from the wild-type gene. The point mutations were then analyzed compared to the restriction enzyme sites of the chb sequence using the NEB cutter, allowing us to examine affected RE sites. Table 1 lists the different point mutations found in the Category 1 chb gene and the restriction site affected by those

mutations. There were 6 particular sites that had substitution at DNA level, of which substitution at nucleotides 2281 -2287 was the most prominent mutation. Mutation g.1956 G>A g.2281_2287 TCCAAGC> ATGTCGT g.2281_2287 TCCAAGC> ATGTCGT RE Site Affected GG^AAT_C CCAAGCTT!GGCAAG^ATGGTTT A^AGCTT Restriction Enzyme Affected HinfI FspEI HindIII

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In order to correctly identify the Category 1 chb gene, RFLP analysis was necessary in the screening process. In RFLP analysis, the DNA is cut up into small fragments using restriction enzymes to produce a large number of short fragments. Therefore, restriction enzymes that will generate fragments that differentiate the mutant gene from the wild-type gene must be selected. This particular screening took advantage of the fact that the EcoRI enzyme cut on both ends of the chb gene sequence, thereby effectively cutting out the entire chb gene sequence out of the genomic DNA. Then, the second enzyme of choice needed to be an enzyme that recognizes a site that changes with the Category 1 mutations. There were several restriction sites that were affected in the mutated chb gene, but HindIII was selected as the optimal restriction enzyme for the double digestion, because 1) its restriction site is clearly disrupted by the large mutation sequence at g.2281_2287, and 2) its restriction site occurs less frequently, producing fragments appropriate for analysis, but not producing too much fragments that makes it difficult for comparison. Section 2: Data Analysis Once the mutation in the Category 1 chb gene was analyzed and restriction enzymes were selected, it had become clear which portion of the chb gene should be used to make the Southern blot probe against. The probe must recognize the DNA fragment that changes between the wildtype and Category 1 mutant double digests. Since the mutation affecting the HindIII restriction site in the Category 1 chb gene occurs at nucleotide 2284, our research team decided to use Primer 770 and Primer 771 for the PCR reaction, which amplifies nucleotides 1964 2695. The PCR reaction was expected to produce a 732 base-pair PCR product. The gel electrophoresis analysis of the gel showed a PCR product band with the appropriate size as in Figure 1 below. The product from this PCR reaction

PCR1 was purified using a gel purification method, analyzed with the Nanodrop Spectrophotometer (A260/280: 1.89) and chemically tagged (DIG-UTP) using the random primed DNA labeling technique with the Klenow DNA polymerase.

Figure 1: Gel Electrophoresis of PCR1 - The PCR1 product using Primers 770 and 771 are shown in the lane (The gel was shared with two other individuals using Lane 5 and Lane 7. This report will focus on the the data obtained by analyzing PCR1 in Lane 3 highlighted in the blue box). The amplified portion is a 732 base-pair region of the chb gene (nucleotide 1964 - 2695). The brightness of the band compared to other students bands suggest difference in DNA amounts loaded. The purified PCR1 yielded a A260/A280 of 1.89.

The screening our research team designed is based on the concept that genetic variation can result in RFLPs and that careful selection of restriction enzymes and probe design can allow RFLPs to be detected by Southern Blot. With selected enzymes EcoRI and HindIII, restriction digests for the RFLP analysis were performed by setting up no-digest / single-digest / double-digests on pBMB, pBMB1 and SDS genomic DNA samples (092308.C6 and 070505.A1). The single and double digests of pBMB and pBMB1 became controls the sample SDS genomic DNA would be compared to. Based on the analysis of the restriction sites of the wildtype and those disrupted on the Category 1 chb gene, it was predicted that the restriction digestion would show a major difference in the double digest reactions: a double digest of PBMB would show four bands of lengths 2686, 1933, 1370, and 350 basepairs, while PBMB1 would show three bands of lengths 2686, 1933, and 1720 base-pairs. This pattern

of bands can be easily visualized by Figure 2a, which shows the rough schematic of plasmids pBMB and pBMB1. Both pBMB and pBMB1 would have the same design as the plasmid in Figure 2a, the only difference being that pBMB1 would have the HindIII at nucleotide 2284 disrupted as indicated in the figure. Figure 2a helps to predict the expected band patterns of the digestion reactions as shown in Figure 2b, which was used to compare the actual gel electrophoresis results to. The gel electrophoresis of the restriction digests showed most of the expected bands as shown below in Figure 2c, 2d, and 2e.

Figure 2a. A Shematic Design/ Representation of Plasmids pBMB and pBMB1 the major difference between pBMB and pBMB1 would be the restriction site HindIII at 2284.

Figure 2b. The Expected Band Patterns of the Digestion Reactions: No digestion of both pBMB and pBMB1 results in a supercoiled DNA band of approximate length of 6kb; single digestion of both pBMB and pBMB1 results in a band corresponding to pUC19 (2686 bp) and the chb gene (3660 bp); the double digests of pBMB and pBMB1 show major differences.

Lane 1: MW Ladder Lane 2: pBMB (Wildtype chb plasmid) no digest Lane 3: pBMB single digest Lane 4: pBMB double digest Lane 5: Blank Lane 6: pBMB1 (Cat. 1 chb plasmid) no digest Lane 7: pBMB1 single digest Lane 8: pBMB1 double digest Lane 9: Blank Lane 10: SDS Genomic DNA (092308.C6) single digest Lane 11: SDS Genomic DNA (092308.C6) double digest Lane 12: SDS Genomic DNA (070505.A1) single digest Lane 13: SDS Genomic DNA (070505.A1) double digest Lane 14: MW Ladder
Figure 2c. Gel Electrophoresis Results of Digestion Reactions: The band patterns are similar to the expected patterns shown in Figure 2b. Lane 2 shows two bands instead of the one expected; this is because the plasmid exists in different forms. The two bands are most likely bands from a nicked plasmid (open circle) and supercoiled / relaxed-supercoiled plasmid that run differently. Lane 4 shows 3 of the 4 expected bands clearly but the 350 bp band does not appear visibly. This is fixed by adjusting the exposure of light as seen in Figure 2d. Lane 6 is missing bands because the well was puncture during the loading step and DNA was lost.

Figure 2d and 2e. The light exposure adjustment shows more clearly the band corresponding to the 350-bp expected band in Lane 4 (Figure 2d). The change in light exposure reveals a more clear separation of the bands (length 1933 bp and 1720 bp) in Lane 8 (Figure 2e)

The PCR product mentioned above (Figure 1) was purified and used as a template to make the probe for the Southern Blot analysis. The probe was chemically labeled with DIG-UTP in a labeling

reaction using a modified DNA polymerase known as the Klenow fragment. The Klenow fragment lacks the 5 !3 exonuclease activity, which is normally involved in the primer removal during DNA replication. The absence of the 5 !3 exonuclease activity allows the labeling reaction to produce small fragments of probes corresponding to a specific region. The restriction digests analyzed by gel electrophoresis (Figure 2c, d, e) were then transferred to a Southern Blot and developed to visualize and detect hybridization of the probe. In the development steps of the Southern Blot, the use of the blocking agent was critical, inhibiting any non-specific binding and excess background signal. The developed Southern Blot membrane is shown in Figure 3. The probe used in the Southern Blot corresponds to a specific region (nucleotide 1964-2695) within the chb gene, and therefore must only detect bands of DNA that contains the chb gene, which are the whole plasmids pBMB and pBMB1, the 3660-basepair-long chb gene itself, the 1370-basepair-long band, 350-basepair-long band, and the 1720-basepair-long band. !
Figure 3. Color-developed Southern Blot Membrane: The chb gene containing fragments are hybridized with the probe and visualized by the color substrate. The red circle is marking a shift of bands that may have been caused during the gel electrophoresis step after restriction digestion. This shift of bands moved the 350-base-pair band slightly upward than where it is originally positioned. Lanes 5 shows bands most likely due to contamination crossed over from Lane 6. Lane 6 also shows some band contamination; the bands corresponding to the 1933-basepair and 1720-basepair bands are likely a result of contamination from Lanes 7 and 8. Lane 13 shows a band at the top that corresponds to the size of the whole pBMB and pBMB1 plasmid (approximately 6kb). This band is visible due to the fact that the digestion was only carried out partially and that undigested genomic DNA remained.

From the analyses of the Category chb gene compared to the wildtype, our research team was able to distinguish Category 1 SDS data in the outbreak.

Part B: Kit Marketing Section 1 Shell disease syndrome is a condition that affects a wide range of freshwater and marine crustaceans. Signs of SDS include erosion and pitting of the exoskeleton, caused by chitin-degradation activities by various microorganisms. Infection is usually limited to the exosksleton, but once the underlying living tissues become infected, it can result in death of the crustacean. SDS occurs relatively rarely in natural populations, but appear more prevalently in captive populations, affecting many aquafarms and marine farms. Chitin is a biopolymer of !-(1,4)-linked N-acetylglucosamine (GlcNAc), and is a major component of the exoskeletons of crustaceans. It is known that chitin degradation occurs only after the death of the crustacean, yet shell disease syndrome causes exoskeletal degradation in living crustaceans. Various bacteria and fungi are thought to be associated with the cause of lesions, one of which is Vibrio Harveyi. The nucleotide sequence of the chb gene encoding the N,Ndiacetylchitobiase, the protein required for chitin degradation, of Vibrio harveyi has been determined (Soto-Gil et al. (1989) The Journal of Biological Chemistry, 264: 14778-14783) and cloned in E.coli and isolated (Jannatipour et al. (1987) Journal of Bacteriology, 169: 3785-3791). Through research and analysis on the wildtype and SDS-infected chb gene, our company was able to develop a SDS screening kit that allows detection of Category 1 SDS isolates based on RFLP analysis. The theory of RFLP (Restriction Fragment Length Polymorphism) analysis takes advantages of differences in DNA between individuals/organisms that result in different fragments when digested with restriction enzymes. RFLPs arise because mutations can create or destroy the sites recognized by specific restriction enzymes, leading to variations between individuals in the length of restriction fragments produced from identical regions of the genome. Differences in the sizes of restriction fragments between individuals can be detected by Southern blotting with a probe specific for a region of DNA known to contain an RFLP. No two individuals have identical DNA, so RFLP analysis allows identification of the patterns that occur in DNA. For example, RFLP analysis of a family can detect the segregation of an RFLP that can be used to test for statistically significant linkage to the allele for an inherited disease or some other human trait of interest. Additionally, RFLP fingerprinting is also used in forensics analysis to identify crime suspects. This kind of DNA fingerprinting is applied to the SDS screening kit, allowing scientists to classify SDS outbreaks by assaying isolated genomic DNA.

Section 2 The rapid screening kit developed by our laboratory research group is useful for detecting Category 1 SDS outbreaks and categorizing unknown samples of SDS. The kit analysis steps include 1) the Restriction Digest Step 2) Gel Eletrophoresis Step, and 3) the Southern Blot step. The kit components include: Kit User Manual o The kit user manual will briefly outline the experimental procedure that needs to be performed in order to correctly analyze the SDS genomic DNA samples. o Step 1: Perform Restriction Digest reactions of control DNA samples and SDS genomic DNA samples using EcoRI and HindIII enzymes in NEBuffer 2. Each sample should go under single and double digests to be analyzed. o Step 2: Perform Gel Eletrophoresis. Run the restriction digest RFLP samples on agarose gel (max. of 125V) until the bands are well separated. o Step 3: Transfer the RFLP-containing gel onto a Nylon membrane in the Southern Blot step. Block the membrane and add the DIG-UTP labeled chb probe to incubate and hybridize overnight. Develop the Southern Blot by adding the color substrate that is acted upon by the alkaline phosphatase enzyme. PBMB and PBMB1 o These two plasmids are used as controls for the restriction digests and Southern Blot development EcoRI and HindIII Restriction Enzymes o Allows restriction digests of the SDS genomic DNA samples along with the control DNA samples NEBuffer 2 o Buffer used for restriction enzymes using EcoRI and HindIII DIG-UTP labeled chb probe o This DIG-labeled probe is hybridized with the target DNA in the Southern Blot step to detect the DNA fragments containing portions of the chb gene Blocking Reagent o Ensures the specificity of the binding of the probe to the sample DNA by blocking the membrane surface and target DNA. Reduces non-specific binding of the probe to the RFLP fragments Anti-digoxigenin/ alkaline phosphatase conjugate o Contains the antibody that reacts with the labeled DNA covalently linked to the enzyme that produces the color reaction NBT/BCIP o Color conjugate substrate of the probe that allows visualization of the Southern Blot Development

Section 3 The SDS Screening kit includes two control DNA samples pBMB and pBMB1 to which SDS genomic DNA samples can be compared. When the restriction digest reactions are set up for these control DNA samples and gel electrophoresis is performed, pBMB and pBMB1 will give distinct bands that will distinguish one from another. Plasmid pBMB has all the EcoRI and HindIII sites intact, and thus in a double digestion reaction, produces 4 distinct bands: the 2.6kb band corresponding to pUC19, a 1933bp band, a 1370bp band, and a 350bp band. pBMB has one of the HindIII disrupted due to mutation, and thus in a double digestion reaction produces 3 distinct bands: the 2.6kb band corresponding to pUC19, a 1933bp band, and a 1720bp band which is a combination of the 1370bp band and 350bp band seen in the double digestion of pBMB1. For single digestion reactions both pBMB and pBMB1 show two distinct bands: the 3.6kb band that corresponds to the chb gene, and the 2.6kb band corresponding to pUC19. This is clearly shown in Figure 4. The bands circled in red are the bands that will show up in the Southern Blot developed with the color substrate. These bands contain portion of the chb gene that can be detected by the probe. The figure below can be used as a reference when performing the screening procedure.
Figure 4: Reference Gel Diagram of the Control DNA Samples pBMB and pBMB1: The control DNA samples show distinct bands that can be distinguished through restriction digestion reactions. The different RFLP fragments can be used as a control reference and compared to the RFLPs produced by the SDS genomic samples.