Difference between revisions of "Team:IIT Kharagpur/Blog"

Food Spoilage ­ What your mom doesn’t know about!

Food spoilage is a common kitchen disaster hiding behind a closed shelf or buried deep within refrigerator which all of us have been frustrated with. It is one of the most important reasons a lot of quality edible food product go to waste. Spoilage is characterized by any change in a food product that renders it unacceptable to the consumer from a sensory point of view. (Lone Gram, 2002) In reality it is a vibrant problem though pictured individually this picture does not zoom out beyond minor negligence in kitchen – leaving yogurt out in open, cauliflowers pushed too far behind in the chill tray. However these food spoilage incidents do happen at industrial and farm level too where a lot of quality raw product can go to waste before it can be made fit for consumption. Expenses which would have to be taken into consideration because of it would include: medical care, including hospital costs; laboratory investigations; the cost to the economy generally of production and wages lost through illness; loss of production and market share for food manufacturers implicated in outbreaks, and legal expenses in the event of prosecution or claims for compensation (Eley, 1992). All of this accounts for a large economic loss.

There are two main reasons for food spoilage: ­ “Naturally occurring biochemical changes” and “Microbial Activity”. Similar to human’s digestion microbes – bacteria and fungi break down the food particles into simple derivatives acids and similar other waste products may be released changing the physical properties and chemical structure of our food. In severe cases they can cause food poisonings and in extreme experiences have also resulted in death! Between 1980­ 1989 Cases of food poisoning bacteria in England and Wales rose from 10856 to 25818. (Eley, 1992).

When food gets spoilt we generally tend to identify it by tasting or detecting change in color, texture or smell. Simple and comfortable these tactics are, they elude us from identifying these issues beyond our daily context. One simple example of this would be biofilm formation in food processing plants where they cause extensive losses due to equipment failure or necessary extensive cleanup.

In past few decades detection of quorum sensing signals in spoiled food products has added a new dimension to study the process of food spoilage. The bacterial phenomenon of cell­to­cell communication using signaling molecules is known as quorum sensing. Bacteria takes decisive action based upon assessing local cell densities. Cell­to­cell communication depends on the production of, secretion of, and response to small, diffusible signal molecules called "autoinducers". The signal molecules are produced and secreted at a basal level during bacterial growth. Their concentration in the environmental medium or matrix increases as the bacterial population expands, and when it reaches a threshold level (quorum level). It induces phenotypic effects by regulating quorum­sensing dependent target gene expression. This phenomenon occurs without any external intervention and is also referred to as auto induction. A very common quorum system found widely in gram negative bacteria is AHL based communication. AHL or N­Acyl Homoserine Lactone is an autoinducer, a part of quorum sensing network in multiple bacteria and has thus confirmed the role of inter cell signaling pathway in food spoilage. They are found in milk and other dairy products which are the fastest among the perishable items to get spoilt. A systematic study has discovered spoilage to be caused by AHL producing bacteria. This study and its results brought new perspective into how food spoilage can be prevented by selectively targeting this signaling pathway and thus preventing microbial action to take place despite their favorable number and conditions.

Quorum­sensing signaling molecules can be detected from cell free supernatants, extracts of food samples, and spent culture supernatants of bacteria isolated from food products (Ammor and others 2008).The use of bacterial biosensors has made the detection and quantification of different types of these signals like AHLs easier, economical, and faster. An AHL biosensors contain a functional LuxR family protein cloned with a cognate target promoter (usually the promoter of the cognate luxI synthase), which up regulates the expression of a reporter gene encoding for a phenotypic response only in the presence of exogenous AHLs, as they do not produce the signaling molecules but only possess their cognate receptors.

Affirmative action using quorum sensing inhibitors can be taken in food preservation also. Quorum­sensing inhibitors are typically analogues of the AHLs, or compounds that degrade AHLs. A promising group of quorum­sensing inhibitors is the halogenated furanones produced by the Australian red alga, Delisea pulchra (Givskov and others 1996). The initial cytotoxicity and chemically unstable nature of these inhibitors has prompted the screening of nontoxic quorum­sensing inhibitors from natural sources. Plants used in traditional medicine are one of the most promising areas in the search for new biologically active compounds. In a study by Vattem and others (2007), dietary phytochemicals from plants known to have several health benefits and antimicrobial activity, exhibited quorum­sensing inhibitory activity at sub lethal concentrations. Vanilla, a widely used spice and flavor, can inhibit bacterial quorum sensing. Quorum­sensing inhibitors may prevent colonization of food surfaces, toxin formation, and proliferation of food­related bacteria. The natural occurrence of quorum­sensing inhibitors is an important consideration for the assessment of their toxicological status and may facilitate their use in food as preservatives and hence pave way for the application of novel food preservation techniques. (Rai, 2011)

Recent modern applications of microbiology in food spoilage have caused much buzz in academic world. The discovery of novel biochemical pathways and subsequent development of their applications is increasingly bringing up new perspective to combat the intimidating challenge of food security. Though the general masses at large is still oblivious to role of microbiota in their dietary habits. Fermentation helps in making bread and pasteurization is enough to purify milk is the saturation of public’s knowledge about food spoilage. A systematic campaign to spread awareness at grass root level would boost public’s interest in food spoilage beyond their monthly budget into the spectrum of global challenge of food security. That would hopefully lead to more developed industrial base providing cheap protection against food spoilage..

Bibliography

Eley, A. (1992). Food Poisoning.

Lone Gram, M. R. (2002). Food spoilage—interactions between food spoilage bacteria.

Rai, A. J. (2011). Bacterial Quorum Sensing and Food Industry.

Team building and motivation:

As this is the first time we, the students of IIT Kharagpur are participating in iGEM, we did not have a pre­existing team of seniors to guide us and make things streamlined for us. Had there been an existing team, we would have had regular rounds of selections into the team and organised way of work right from the beginning. But this was not the case with us. The students in our department were not aware of iGEM and so it was not easy to straight­away build a team.

Few of our seniors, Krishna, Madhur, Kaustav and Ayushman, students in the current fifth year batch, took the initiative. During their summer interns in various universities across the globe, they had come across iGEM teams operating in the respective institutions. Gradually, they came to know about iGEM in greater detail and the opportunity we had as a commendable participant in the competition. On their return to Kharagpur, they called in all the interested students from the third and fourth year batches and held regular meetings to discuss about iGEM and explain its significance. Students, who had poor background on synthetic biology were given articles to read so that they cleared their funda. Finally, after a few weeks, towards the end of January, we had a fairly motivated team ready to brainstorm ideas for the much coveted competition.

Formulation & finalization of idea:

For facilitating the idea formulation process, the entire team of third years and fourth years was divided into five sub­teams, each composed of 1­2 fourth years and 2­3 third years. Each team was allotted the task of going through literature and coming up with new ideas that could act as potential bases for our iGEM project. Each team used to discuss independently, meet frequently in the library, and prepare presentations on the idea that they thought upon. As the exams were approaching and the deadlines were less than 2 weeks, the teams had to go through a really rough patch. Some members were not able to come regularly due to some urgent problems and the other members used to take up their job and do it religiously. It was real teamwork at display then.

On the day of the idea discussions, each team presented their idea before the fifth years and other teams. It was subjected to rigorous scrutiny and a torrent of questions basing on which the ideas were evaluated. Out of the five ideas, two ideas were eventually shortlisted and the entire team was now re­grouped into two sub­teams working independently on the two ideas. The existing knowledge from the previous level was augmented with in­depth book­reading and feasibility analysis. After a week, the two ideas were consolidated and extended presentations were made to be presented to the professors.

Pitching of idea in front of professors:

The shortlisted ideas were presented in the form of two long presentations, followed by a detailed question­answer session. All faculty members of our department were present and were actively involved in it, putting forth their ideas and queries. After a 2 hour­long debate, finally one of the ideas was chosen to be feasible for the project. However, due to lack of any available prototype at that point of time, many of the professors rejected the idea of participation in this year’s competition, stating that it would be unwise to give consent to something which had no proof of materialising within a week’s time. As the deadlines for registration were approaching, we had serious troubles in convincing the professors that we would be able to deliver the results in the right time.

This session was to be followed by a meeting with the dean and a panel of high­ranked officials of our institute. But that too got delayed due to the absence of the dean on some urgent matter. We had no option in hand but to request the iGEM office for an extension in the deadline on the plea of delay in arrangement of funds. To our luck, the iGEM office responded positively, and extended the deadline by a few weeks.

Registration, consolidation of idea and selection of guide:

Not all the professors had opposed to the idea of our participation in this year’s iGEM. Prof. Dey and Prof. Ganguly were in strong favour of the idea. This prevented us from losing all hope and we kept on the struggle. With their help, we were able to rearrange the meet with the dean and after a lot of effort, the plan was approved. As a result, funds were sanctioned by the institute and we were able to register towards the end of April. All the registration formalities were carried out and Prof. Ganguly was chosen as the principal guide of our iGEM team because of his expertise in synthetic biology and his willingness to dedicate time and resources for the consolidation and success of the project.

In the next few weeks, several meetings were held amongst the team members and with our guide. Related papers were presented and discussed and the idea was consolidated. Accordingly, the work­flow for the project was chalked out. As many of the fourth year students were going abroad for summer internships, the ones staying back had to schedule their work in the lab and distribute the work to the many third years who were staying back too. Those members who were going for interns also ensured progress of the wiki content development and other non lab­related assignments.

Lab work:

Wet­Lab work regarding transformations and cloning had started before the arrival of the Kit of Parts. Sriram and few other third year students had stayed back in the summers and practised the various methods of rDNA technology. Prof. Ganguly, along with few of his PhD students, was himself present throughout the summer to train our team members in these procedures. The Kit of Parts arrived in the summer vacation period itself. After that, experiments began with full force and as the vacations ended, the rest of the team members, who had gone for their internships, joined in one after the other. All the wet­lab work was carried out in the fully equipped UG lab of our department which remains open 24X7.

Our experiments were often interrupted by setbacks and challenges. When we first tried to transform the luxR and crtEBI, we did not get the colonies. This may have been a result of experimental errors like improper spreading or so. However, after several attempts, we could rectify it and obtained colonies. We checked our plasmids by using appropriate restriction enzymes and running gels. Another serious problem arose in case of ligation. The ligation was carried out for 16hrs with an insert:vector ratio of 3:1 but we did not get any colonies at all! We assumed that ligation was not occurring due to improper digestions of the insert. So we carried out sequential digestion and again tried the ligation in the same procedure. This time we got a single colony and on screening it, we found it to be the undigested luxPR transformant! We were terribly disappointed.

Next, we tried checking whether there was any problem in the competent cells. For this, we took a positive control of 50ug/ml of luxPR DNA and tried the ligation procedure again but with no better result. After that, we started working on another way of cloning where we tried to get 55 bps of luxPR cut with restriction enzymes. On performing sequential digestion of luxPR and crtEBI, and running the product on 2% agarose gel, we were not able to get the 55 bps band (instead, we got a gradient of bands of same length). Yet, we did not lose hope. We started again with the “3A Assembly” : sequential digestion of luxPR and crtEBI, PCR purification, double digestion ­ luxPR with ECoRI and crtEBI with XbaI followed by heat kill for 5 mins. This time we used 3:1:0.5 , 1:1:0.5 of luxPR:crtEBI:kanamycin backbone DNAs for ligation (16hrs) and finally succeeded in getting colonies.

Several other hurdles were also encountered by our wet­lab team. But our motivation boosted our patience and perseverance to keep on struggling till we achieve the result.

National Meet­up:

The IISER Pune iGEM team organized an Indian iGEM meetup during July 16­17, 2015. This meetup had three of the four Indian iGEM teams (for the year 2015) participate in it, namely IIT Delhi, IISER Pune and IIT Kharagpur. IIT Delhi had sent three representatives of their team, while two of our team members ­ Harsheel and Rhushikesh represented our team there. During the first day (16th July), there was an informal discussion session amongst all the teams, followed by IISER Pune’s project presentation in the afternoon. While the second day (17th July) had the presentations of our team (in the morning) and IIT Delhi’s presentation in the afternoon.

IISER Pune made a really comprehensive presentation, which left the entire audience in awe, and in a way left the other teams with no choice but to further improvise their presentations. All the IISER Pune team members participated and contributed to their extensive presentation, which had a significant amount of their project modelling data as well (computer simulation based work). They had also covered good ground with regard to their wet lab work, and had it divided into three sub­parts, each handling a particular aspect of the wet­lab work. While IISER Pune wishes to compete in the Health and Medicine track of iGEM, IIT Delhi wishes to compete in the environment track. IIT Delhi also made a very descriptive presentation of their idea, which was appreciated by the audience. After each of the team presentations (which typically lasted about an hour), there were question­answer sessions held, where the clarity and idea of each of the teams’ projects were scrutinized.

Very useful suggestions and points were raised for each of the teams post their presentations, and all the discussions (both formal and informal) were very productive and propitious for the teams, in general. We learnt a lot about iGEM through this meetup, got to meet a lot of people, and are confident that the advice and suggestions given during this meetup would definitely help us give this first attempt at iGEM our best shot.

NCTU­Formosa regional meet up:

The NCTU Formosa iGEM Meetup was held during July 19­23, 2015. This was the third time NCTU organized its own meetup, and this time, it kept it open for all the Asian iGEM teams. It was organized on a pretty grand scale with 30 teams (from countries like Taiwan, China, India, etc) participating in it. The two Indian teams participating in this meetup were IISER Pune and IIT Kharagpur. The option was given to teams to either go and attend the meetup in person, or to present their ideas via video conferencing (Skype). Accordingly, our team went ahead with the second option, and was allocated a video conference slot for the evening (Taiwan Time) of the third day of the NCTU Meetup (21st July).

The main idea behind our team’s participation in this meetup was to get advice and suggestions from experienced iGEM teams with regard to our project ­ wet lab, policy and practices (social outreach, meetups, collaborations, lab visits), modelling and so on. The format of the online conference was 20 minutes for the presentation and 20 minutes for the question and answer session. Harsheel, one of the senior members of our team gave the presentation of our project idea, and the questions that followed (which were pitched by the audience gathered at the seminar room in NCTU, Taiwan) were tackled by some of the senior members of the team, as a whole. Very valid questions were raised by the audience, and many useful suggestions were given with regard to how we could improvise our presentation for the Giant Jamboree, and tackle each of the factors (taken into account by the iGEM judges), so as to give our first time participation in iGEM the best we have. This meetup was indeed a great learning experience, and has geared us up for the grand finale at Boston!

Strategy discussions:

After our institute re­opened post summer vacation, we have had a series of meetings amongst the team members. They were mostly aimed at focusing on the design of wiki and all other stuff regarding meeting the necessary requirements for a proper website. Apart from that, discussions on the design of poster, banner and video were also held. Proposals for community participation were also entertained.

On the 3rd of August, our team had a major strategy discussion meeting covering all the pressing issues with regard to the pending work for the grand competition finale at Boston.The entire team actively participated and contributed to the discussion, several beneficial suggestions were put forth, and updates on all the completed tasks were made. It was definitely a very productive session, and tasks were assigned hoping to execute our re­defined strategies in the best way possible!

On the 15th of August, some of our team members met for the iGEM project modelling related discussion.Equations hypothesizing the reaction rate kinetics of our system were put forth, and the required biochemical assays for each of the entities were discussed. The kinds of graphs to be made and associated modelling to be done were finalized.Post all the technical discussion, we ended the session on a lighter note by deciding a catchy tag­line for our project! On the 22nd of August, we had a meeting regarding sponsorship and media outreach. We discussed important methods to be undertaken to increase awareness of our project. An article was written by Jugal to be sent for media purposes and we also put forth ideas for the making of a brochure.