Abstract In food industry such as the production of cheese, bacteria takes a significant role on the wide variety of cheese. Lactic acid bacteria(LAB), a bacteria that can be found in the production of cheese, its stress gene was investigated in the experiment by using various biochemical and genetic techniques to identify and extract. The characterisation of the strain illustrates how identification of strains differ using different methods, such as gram stain and 16s rRNA screening. After the characterisation, the stress gene isolation assist the further understanding of the gene on LAB be giving different stress in future work.
Aims Whole experiment can be separated into two parts (I) The characterisation of the isolate(lactococcal species) given by * Using simple biochemical tests * Using genetic tests by extraction of total genomic DNA * Amplifying the gene encoding for the 16s rRNA by PCR * Understanding the fermentation growth kinetics in controlled liquid media to know the capability of organisms * The investigation of the digestion of the 16s rDNA amplicon with Hinf1 on 16s rDNA strains (II) The amplification and cloning of a specific stress gene given by * Designing primers for cloning cloning stress gene into cloning vector (pGEM-T) * cloning stress gene into expression plasmid(pET23b) – not done Literature Review Lactic acid bacteria (LAB) LAB are defined as bacteria that produce lactic acid as their major fermentation production. The examples of LAB include Streptococcus, Enterococcus, Leuconostoc and Lactococcus. They are gram-positive rods or cocci bacteria with high AT content. (Prescott et. al, 2008). There are few applications of LAB in food, medical and biotechnological industries. The major use of LAB is on the food industries. The non-pathogenic species, hich helps the process of fermentation of different food such as dairy products, meat and vegetables. They mainly convert the hexose sugar exist in food to lactic acid( Makarova et al. , 2007). Recent research shows that they can produce antimicrobial compounds that have probiotic properties, they also helps different medical aspects. It includes “antitumoral activity, reduction of serum colesterol, alleviation of lactose intolerance, stimulation of the immune system, stabilization of gut microflora”(Faid et. al, 2009). Another application in medical field is the production of bacteriocins by LAB.
A more well known one is Nisin, which is a lantibiotic produced by Lactococus lactis. (Anastasiadou et. al, 2009) Different examples are further illustrated in following paragraphs indicating the industrial importance. Food Production Feta Cheese, which is a Greek cheese traditionally made from raw milk without starter cultures. The contemporary production is in well-structured dairies. Ewe milk is thermized(treated at 63 to 65°C for 16 s)( GRIFFITHS, 1998) with yogurt as starter. From Feta Cheese, different species of LAB can be identified, they are responsible for the unique flavour of Feta cheese. Arvanitis et. al, 2009). LAB can also be used to improve the preservation of ready-to-eat(RTE) seafood such as cooked and peeled tropical shrimp and cold-smoked salmon. France is the second largest shrimp exporter in Europe and largest salmon exporter in the world. Technology for preservation is significant for them. Chemicals such as nitrates, sulfates and sorbate can be added to preserve shrimp in France. However, these chemicals are forbidden for smoked salmon. Under these circumstance, biopreservation plays a significant role for smoke salmon industries. (Cardinal et al. 2009) According to a review from Rodgers, “Biopreservation is a preservative method that uses the growth of selected microorganisms on a food product to control the undesirable (spoiling and pathogenic) microflora”(Rodgers, et al. 2003) There are few pathogens such as Vibrio spp. , Salmonella spp. and Staphylococcus spp. can be found in RTE food. Although some of the LAB, such as Lactobacillus sakei or Brochothrix thermosphacta , can lead to spoilage in pure culture, some other LAB species still plays a magnificent role in food preservation. Four strains of LAB show the best result(two Leuconostoc gelidum and two Lactococcus piscium train) in an experiment which can reduce the amount of Listeria monocytogens and S. aureus for up to four weeks. (Cardinal, 2009) Bacteriocins production Bacteriocins are synthesized by ribosome. There are four classes of bacteriocins produced by LAB: Lantibiotics(Class I) which are modified, nonlantibiotics(Class II) which are heat-stable and unmodified (Class III) which are large heat-liable bacteriocins and (Class IV) which are complex molecules with lipid and carbohydrates moieties, As mentioned, chemical preservatives were criticized to be unsafe and the public prefers a more natural way for preservation.
Different classes of bacteriocins have different activity against different species. For example, nonlantibiotics are active against Listeria and Clostridium spp. They work by permealize the cytoplasmic membrane by inserting the C-terminal regions into the membrane through pore formation. Bacteriocin producing LAB can perform a milder and less expensive method to overcome the whole preservation process, while some enzymes can make the pathogens lyses , yet they are more expensive and harder to be controlled. .(Anastasiadou et. al, 2009) Medical Use
LAB can be used as probiotics as well, one of the examples is the compensation of lactase insufficiency. Lactose intolerance means that the concentration of lactase in the small intestine is not enough to digest lactose properly. LAB have lactase activities, when body consumes it, some of the LAB probiotics can resist the bile salt and the pH of the small intestine helping to reduce the lactose intolerance symptoms such as hydrogen exhalation. (De Vrese, et. al, 2001). Overview LAB has significant commercial value in market mainly on food industries.
Its main effects are adding health effect on dairy products, assisting the preservation process. It also helps to make cheese have varies flavor acting as the starting culture. For many countries, instead of using chemical preservatives, LAB is used as biopreservative that gives consumer a more natural image. Throughout the process of product innovation, stress gene plays an important role. For example, for the production of cheese, it can pass many lab tests. When it is transferred to the real world, the stress ene within the bacteria might affect the cheese taste by the impact of the transportation, storage, fluctuation of the temperature, humidity. Knowing stress gene can help for the controlling of product quality. Identification of a stress related gene from Lactic acid bacteria In the whole experiments, stress related gene was chosen and identified from lactic acid bacteria (LAB). Stress, in microorganisms, basically means that some change of the environment that might lead to the death or inhibition of that organism. There are different kinds of stress such as temperature, pH, and oxygen level. Vorob’eva, 2003) The study of stress gene of LAB helps to understand what industrial process can be advocated or avoided to optimize the production and minimize the cost. The oxidative stress for Lactococcus lactis was planned to be studied. It is widely used for food production including the production of Cheddar cheese. During the normal production process, it is not strictly anaerobic. It still involves the contact with oxygen. LAB use oxygen to regenerate NAD+ . In the whole process of converting, there will be an increase of hydrogen peroxide.
Hydrogen peroxide inhibits the growth of LAB. LAB can deal with these hydrogen peroxide molecules by using superoxide dismutase (Sod). During most of the LAB production, there will be an increase in the amount of Lactic acid and thus lowering the pH. In this experiment, the sodA gene would be cloned and studied. It would be tested to justify the effect of concentration of hydrogen peroxide on the production of Sod. (Haandrikman et al. , 1995). This gene will be tested with the difference under aerobic and anaerobic conditions.
The gene will be identified first by giving some specific conditions, in this case, the oxygen, to check if the Sod is present, The “Stress Response Testing” in lab book given should be used. Then use a restrictive enzyme to extract the piece that is responsible for the characteristics. Then use the same restriction enzyme to open the plasmid at the same place in a Vector such as from E. Coli. The gene Sod can then be transferred and have further investigation. (Remark:While in actual experiment, RecA was cloned and extracted instead of SodA gene) Flow diagram Whole experiment can be seperated into two parts 1) Characterisation Fig 1. Basic biochemical tests such as catalase test and gram stain are done to identify the strain of bacteria being investigated (2) Extraction of Stress gene Fig 2. Primers are ligated into pGem-T(cloning vector) in order to be replicated inside a bacterial cell(JM109), the Blue/white selection is to indicate if the ligation is successful. Materials and Methods Gram stain: details refer to A. 7 Pg 58 of the lab manual (Cail et. al, 2009) Gram stain is based on the physical and biochemical properties of the cell wall. It can be divided into gram positive and negative.
Catalase and Oxidase test: refer to A. 8 of the lab report (Cail et. al,2009) Catalase test Hydrogen peroxide is used to test if there is an oxygen production Oxidase test An intense deep-purple hue can be seen within 5-10 seconds if it is a positive reaction. PCR It is used for amplification of primers in the experiment. Amplicon purification Clean up procedure: refer to promega website PCR and Amplicon digestion with HinfI restriction Endonuclease: refer to Pg. 74-75 (Cail et al. , 2009) Results (The result is presented according the flowchart) Biochemical Tests Table 1. howing the result of different bacteria Bacteria| Biochemical tests| Gram stain| | Oxidase| Catalase| | Strain 109| Postive| Positive| Gram positive cocci| Vibrio parahaenopillus| Postivie| Positive| Negative| Enterobacteria aerogenes| Negative| Positive| Negative| Staphylococcus aureus | Negative| Positive| Negative| Lactobacillus brevis| Negative| Negative| Negative| Electrophoresis of 16s rDNA PCR Lactis By visual estimation, for lane1. 3, the size of the band is in between 1- 1. 5 kb Fig. 3 Gel picture of lactis 16s rDNA PCR strains Fermentation result Amount of Lactate produced= (alk used*alk concn* 90. 8) / Fermenter vol. = 41. 00 0. 38/(50/1000)*90. 08 =55. 4ml Digestion of the 16s rDNA amplicon with Hinf1 on strain 1-6 The calculations below is done by the using the lane 2 and lane 3 Fig. 4 Gel picture of HinfI digestion Table 2. marker bp with the distance migrated showed in mm. Marker b. p| Distance migrated by marker (mm)| log10bp| 10000| 7| 4. 0000| 3000| 18| 3. 4771| 1500| 29| 3. 1761| 1200| 35| 3. 0792| 1000| 40| 3. 0000| 800| 46| 2. 9031| 700| 50| 2. 8451| 600| 53| 2. 7782| 500| 58| 2. 6990| 400| 64| 2. 6021| 300| 70| 2. 4771| 200| 78| 2. 3010| 100| 90| 2. 0000| Fig. Standard curve showing the log10bp against distance migrated for lane 14 The standard curve formula: y=-0. 021x+3. 919 Table 3. Showing the distance measured for the bandings for lane 3 (strn 109) with the comparison of the theoretical bp # using first one as an example, substituting x=45mm into y=-0. 021x+3. 919, y=2. 974 by inverse log of 2. 974, 942 is the bp * It is obtained from NCBI website referring to the instruction from week 6 tut. notes Measured distance(mm)| log10bp#| bp| Theoretical bp*| 45| 2. 974| 942| 890| 77| 2. 302| 200| 241| 82| 2. 197| 157| 204| 89| 2. 05| 112| 128| 93| 1. 966| 92| 85|
From the results, the experimental value doesn’t vary a lot from the theoretical value, It is quite accurate since there is only 50bp different in average from band 1-5 with the theoretical value. The difference might be due to contamination of the DNA. For example, at lane 2, it is noticed that there is a smear around it, it means the there are smaller DNA fragments even it’s not cut. The possible reasons for the smaller DNA might be due to the shearing of the DNA sample. Another possible reason is the contamination of the mixture mix, it is because all the lanes (4,6,8,10,12) have significant smear.
There are different digestive sites in a bacteria gene. In this case the section of the Lactococcus lactis has five HinfI digestive sites, they allow HinFI digestive enzyme to digest at those specific positions. Different sizes fragments are produced in the experiment. And as the position of the HinfI sites are the same, it is identified that the strains are from Lactococcus lactis Primer design F primer: CAT ATG ATG CAA AAT GAG TTG CAA GTT T R primer: CTC GAG TTA ATC TTG AAG TTC AAG TCG ATT C Fig. 6 Gel picture showing primers PCR products The band shows that the primers are amplified successfully
Nanodrop Table 4. Table showing the purity of the plasmid extracted It can be seen the purity of the plasmid extracted is high Sample ID| Date| ng/ul| A260| A280| 260/280| 260/230| WA1. 3| 21/05/2009 | 40. 12| 0. 802| 0. 474| 1. 69| 1. 63| WB1. 3| 21/05/2009 | 55. 68| 1. 114| 0. 629| 1. 77| 1. 76| Fig. 7 Gel picture of the plasmid extracted(lane 7,8,9) It can be seen that the band fade off which indicates that the plasmid extracted is too small for significant detection Sequence analysis By using FinchTV, it doesn’t show any sequence, there is no stress gene extracted Example sequence from WebCT vista
Accession: Y17216. 1 Description: Lactococcus lactis cspC and cspD genes Max Ident: 97% There is a 97% similarity to Lactococcus lactis Discussion Characterisation of the strain Traditionally, strains can be identified by biochemical tests such as gram stain, catalase test and oxidase test. However, the tradition phenotypic identification has few disadvantages,(1) It involves the plating of the strains which takes a long time of incubation (2) The judgement is subjective (3) It can not identify the strain specifically(4) the phenotypic way fails when there is a large collections of isolates. (Bollet et. l, 2000)In the experiment, biochemical method was first used to test strain 109, the result cannot show what the strain is. More specific test on 16s rDNA is done. The HinFI site was digested using specific restrictive enzyme, the bands size produced, when comparing with database from NCBI can show specifically which strain it belongs to. As part of the characterisation, the fermentation was done, different LAB has different lactic acid producing rate. By comparing the different lactate production rate, the strains can be identified. Extraction of stress gene Design of primers for stress gene
The significance of the designing of primers is that it controls the stress gene that is going to be cloned. It makes sure that it starts away from the site that can be cloned for effective cloning of the targeted gene. The primers can also undertake the changing PCR temperature. Reasons for blue/white and ampicillin resistant selection The blue/white selection can figure out whether the ligation is successful. The transformates are first plated on X-gal and Ampicillin containing plate. Within the pGem- T easy vector, there is a section of LacZ. LacZ operon is responsible for the production of ? galactosidase that is essential to break down X-gal. When lacZ is not disrupted, blue colonies are formed after the breaking down of X-gal. It contains multiple cloning site(MCS) which allows many different restriction enzyme for digestion. Insertion of the gene is done here. When the lacZ operon is disrupted, X-gal can’t be metabolised and white colonies are formed. These are the successful ligated vectors. (Promega,1998)Since Ampicillin resistant site has restriction sites as well, in order to check if the stress genes wrongly insert, if it is wrongly inserted, then the colony can’t survive.
Through these two selection, it can make sure that the colonies selected have the stress gene inserted at correct site. Sequence analysis The result from the experiment is not successful, there were no sequence found at the plasmid extracted. The reasons might be because (1)The plasmid extracted is too small(2) The concentration of the plasmid is too low for sequencing (3) Unstable temperature throughout the whole experiment . Purity won’t be a reason since the nanadrop step of the experiment shows that the purity is high. (absorbance is 1. when the DNA is pure when the result shows 1. 69 and 1. 77 ) Since the RecA sequence didn’t work out properly. Example from Webct vista is used for illustration of sequence analysis. From the NCBI website, it was found that there is a 97% of similarities of the example sequence to Lactococcus lactis. There was a 3 % difference, it might due to the random mutation in the strain during experiment or site directed mutagenesis. Probably there is a section of the strain that has structure that can complementarily attach the primers. This leads a base mutation in the strand. Future work
In the experiment performed, the stress gene was not extracted successfully, so it should be performed again with more PCR cycles, which can lead to a higher concentration of the primers. Higher concentration of primers can increase the amount of stress gene cloned indirectly. After the successful cloning of the targeted stress gene, it can further be investigated by using the flowchart fig. 8 . By digesting the pGem-T vector which contain the targeted stress gene at site NdelI and NotI and the same sites at expression vector (pET-23b), the cut out stress gene can be ligated to pET-23b. Expression vectors are designed to provide a high level, controlled expression of a target gene with resulting production of a protein product at concentrations as high as 40% of total cellular protein” ( Harwood , 2006). After the transformation into BL21 strain of E. Coli, it can be plated on ampicillin containing plate to have Amp resistance colonies. IPTG can then be added to induce the protein production. The protein produced can be purified and have further investigation of the functions. Few other future works that can be done is to test the stress gene to see the endurance of functioning.
For RecA, the temperature sensitivity can be tested with changing temperature to observe which temperature works the best. Comparisons of different stress gene comparing with RecA can also be done as well. Fig . 8 FlowChart of future work from the point of plasmid extraction Reference 1. Anastasiadou S. , Papagianni M. , 2009. Pediocins:The bacteriocins of Pediococci. Sources, production, properties and applications. Micriobial Cell Factories. 8:3 2. Arvanitis L, Litopoulou E. , Nikolaou S. , Psoni L. , Tzanetakis N and Vassiliadis A. ,2009.
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