Saturday, July 12, 2008

Week 3 SIP - Pilot Food Catering Tech Plant and my beloved MP

Heya guys, hope you all have been enjoying your SIP because i have. I have been attached to the Pilot Food Catering Plant and have started my SIP this week. I am sorry i cannot share any recipes with you guys because it is a trade secret .

There are many jobs to do in a food technology plant. You will have to help the chefs prepare ingredients, perform baking of pastries, grinding of cashew nuts, labeling of food containers, sieving of flour, cracking off eggs, packaging of cakes, maintenance of machines, cleaning the facilities and greasing the baking trays. And this is only the 1st week of my attachment there!!

The preparation of ingredients is very important in baking a premium pastry or cake. Hence it is very imporant to get your measurement units right. A kilogram does not equate to a gram of sugar. Imagine adding a kilogram of sugar into your pastry... YUmYUm.

After the baking proccess and the finished product is achieved(hopefully it turns out well), the TSO and i will have a food tasting session. We will sample the quality of the food and a checklist will be evalutaed. The checklist consist of gradings from 1 to 5 and is very important to gauge the quality of the food before it can be moved to Bristol to be sold. These checklists will not be only given to us, but also to the rest of the students from the Food science and culinery courses for their remarks. Basically the checklists will ask questions on the texture (soft, hard), taste (sour, bitter, sweet, sour), colour (pale, dark, brown), enjoyment (enjoyed eating, feel like puking) and further remarks on how i can be improved. All the checklists wil be gathered and keyed into the computer (not LIS) and tabulated using Excel to find the average response to the food. If the average pertains to liking the product (majority), the cakes/pastrieswill be packaged and labeled before shipped across to the Bristol to be sold.

My Mp involes developing a protocl for zymogram and i briefly explain the principles of a zymogram.

Zymography is a type of electrophoresis technique using SDS-PAGE. It is different from the usual electrophoresis, as it is copolymerised with a substrate (usually geltain or caesin) Why this 2 substrates? Most proteases and periplamic proteins are able to digest there 2 substrates and this allows us to measure and detect the enzyme activity. Another difference for zymogram and normal electrophoresis is that the sample buffer is prepared without boiling and adding of reducing agent such as B-mecapethanol. This is because we do not want to denature the protease and periplasmic proteins as we want them to breakdown the substrate to detct the enzyme activity. After electrophoresis, Triton X-100 is added for the renaturation process and incubated in the digestio buffer for the reaction to take place.Later the zymogram is stained with coomassie blue stain and will show up as clearings/halos whereby there is digestion of periplasmic proteins to the substrate have taken place.

Thanks for reading my entry, have a great MP/SIP ahead

From: Benjamin MA
Class: TG01
Date: 12/07/08


Fluid collectors said...

oh my... sounds like a lot of food is being eaten?

Anw, for Zymographym, it is just to detect if periplasmic proteins are being digested to the substrate? Is it use for other purposes?

And what are periplasmic proteins? Is it different from the normal proteins we know of?

Thanks =).

-Li Ping-
TG o2

tg01 group 2 said...

Hello Li Ping

The zymogram is to detect protease activity. Periplasmic proteins although it is a protein but if u remember from Bpharm, there are lots of signaling molecule such as in the tyrosine kinase pathway, there are protease enzyme activity present and the zymogram can help us detect that. These protease activity occurs in the periplasmic proteins of the S.maltophilia. The protease present will digest the substrate and not the periplasmic proteins digested to substrates. We will den the gel stain with coomassie blue to identify the regions digested and excise protein of spots of interest and analyze them.

Periplasmic proteins are proteins found in between the plasma membrane and the outer cell wall of gram negative bacteria. Yes it is different from other proteins as it can be considered a viruence factor for S.maltophilia used for attachment to host cells.

De Incredibles said...
This comment has been removed by the author.
De Incredibles said...

hi benjamin,
I can't really relate why u're in a pilot food catering tech plant..tot u were attached to the sch lab. but it seems quite good there. =) lols.
Is the designing of the protocol u mentioned part of the periplasmic protein project? How is it related?

Jean Leong

group1 said...

wow! amazing, we're in biomedical science and you're actualy attached to something for food science? hahas! if i'm not wrong, you're doing research and not clinical lab right?

there is something i wondered. can you relate what we learnt in our 3 years and applied in what you are doing now or are you learning totally new things?

hmmmm, being a mountain turtle, can i know why do you want to know if the protein is digested? it is going to do any help to the organisation you're atached to?

Chew Yu Mei

tg01 group 2 said...

Hello Jean

Yes the designing of the zymogram protocol is of my two objectvies for my major project. This is because our school does not have a zymogram protocol and thus in Mole Bio u do not actually see us do zymogram gel electrophoresis :). So if my design of the zymogram protocol is a success (hopefully), it can be used for the school lab for future practical exercise for students in the future.

The zymogram protocol will be important in the analysis of protease activity occuring within the periplasmic proteins. One of the aim is to see the differences in protease activity between 28 degree Celsius and 37 degree Celsius, hence we will run gel electrophoresis using zymogram with protease obtained from the bacteria at these 2 temperatures. As the protease will digest the substrate gelatin, we can eventually see the protein spots or halo in gel. For example, if at 27 degree Celsius we see more halos, that means the protease activity occuring within the periplasmic proteins is greater than that at 37 degree Celsius due to increase digestion of the protease to the substrates.

Thanks for your questions

group1 said...

Hello Benjamin (:
I find it rather weird that how come you are in this Catering Tech plant for your SIP?! haha, hopefully you are enjoying yourself!

You mentioned, Zymography and I want to know what is this usually used for in diagnosis of what kind of illness or diseases? I understand that it is mainly used to detect the presence of periplasmic proteins, but is it used for detecting any illnesses?

-Yvonne Teo

tg01 group 2 said...

Hello Yu Mei,

Yea im quite surprised too lol, im doing research btw. And i cant understand why im doing proteomics (analysis of proteins from bacteria) and Food preparation at the same time ... Speak about hygiene =.=''

From our 3 years, the things most applicable to my project now are stuff learned from our Biochemistry, Molecular Genetics and Molecular biology and a little bit on basic microbiology. Gel electrophoresis is an extremely big part of what im currently doing such as i must know what are the functions of bisacrylamide, agarose gel, SDS, buffer systems and such. Im also learning how to streak bacteria properly, how to inoculate them, how to perform bradford assays,, how to use microsoft Excel and how to perfrom chloroform shock. As for the SIP part (food), yeah it is totally new to me. BTW i havent learnt how to cook before so this is entirely new experience for me.

Er let me briefly explain what are the parts or procedures in a zymogram.

1st your would have to have a substrate (gelatin which im using) copolymerized into the gel. Thereafter you would either run a 1D gel electrophoresis or 2D gel electrophoresis after loading your sample in. During electrophoresis, the protease will be seperated based on difference in charge due to SDS 1.4gSDS /protein or isoelectic point and molecular weight in 2D gel electrophoresis. After the proteases are seperated on the gel, Triton X or renaturing buffer is added to renature the protease. A developing buffer will be added to allow conditions optimal for the protease to start digesting the substrate gelatin present in the gel. The gel is later stained with coomassie brilliant blue to identify the area in the gel digested and analysed.

im sorry i cannot reveal anymore on the protocol or im going to get it from my supervisor. LOL! i hope i have enhanced your understanding on what is a zymogram.

Thanks for your questions!

tg01 group 2 said...

Hello Yvonne

Er zymography is not used for detecting or identification of any illnesses. It is used to characterise and identify proteins based on proteins spots/halos (clearings) r bands that have appeared on the gel after electrophoresis. After the analysis of the clearings, we have a better understanding on the protease acitivity present. As little is still known on the virulence factors of S.maltohpilia, we do not know whether these proteases actually can confer pathogenicity to the bacteria. It Mat or may not. Hence we would like to identify the protease present and this is done by genetic typing of the protease and comparing it to the genome library for genetic sequencing (this is not my project scope). After confirming the protease present, it (protease)will be related to well- bacteria such as S. aureus on the same kind of proteases (such as maybe collagenase is present on periplsmic proteins of S.malotphilia), hence we can later on confirm whether these proteases are pathogenic or not. And drugs can be developed to target these proteases and the periplasmic proteins.

MadTechs said...

Woah, two continuous entries! Sounds like your SIP is going to be a yummy experience!

Anyway, just wanted to know:

1) What is the MoA of B- mecapethanol as a reducing agent? (I thought the spelling of B-mecapethanol is actually B-mercaptoethanol? Or is it a different substance?)

2) What is the MoA of Triton X-100 in the renaturation process?

MoA = Mechanism of Action

Thanks a million!
Quan Jun
Group 08
Posted: 14 July 2008

tg01 group 2 said...

Hello Quan Jun

Sorry for the spelling, it should be B-mercaptoethanol. B-mercaptoethanol works by breaking/cleave the disulfide bonds of the protein, thus disrupting the proteins teriary and secondary structures.(linearised/denaure proteins)

Fluid collectors said...

hi ben, what are the different kind of enzymes that will digest the substrates? can you state a few examples?

tg01 group 2 said...

Hello Yu Xuan

YOz... very good quesiton my purpose of this project is to find out this lol.... so i dont know the answer now. Ill reply u in the future when ive characterized and identified the protease

But i suspect it is the beta lactamase present in the periplasm. This helps break down the antibiotics so that s.maltophilia is resistant to a wide array of antibiotics.

I hope ive answered your question...

De Incredibles said...

Hi benjamin,
What do you get from the pilot food catering tech plant for the periplasmic protein project? they have a zymogram protocol there that can be revised or sth?

Jean Leong

tg01 group 2 said...

Hello Jean

The food catering tech and the zymogram is 2 different matters at 2 different locations in the school.

In the food catering tech, my SIP is performed there, whih is totally unrelated to my MP. My SIp mainly involves baking, preparation of ingredients for the chef and basically perform duties expected of a technical support officer in a lab.

For my MP, it is done in the AS 4 research lab. The subject is on zymogram and the development of the 2d zymogram protocol. I would have to perform research on 2d zymogram protcols that were performed in other experiments in the pastand based on the steps in the protcols found relevant, it would be used to help me construct a protocol for 2d zymogram. I would den have to test out this protocol to get valid results.

From: Ben

De Incredibles said...

hi Benjamin,
Thanks for clarifying my doubts. Hahas. sry for the misunderstanding abt the SIP and MP and thus all the 'weird' qns I had. Tk care.thanks alot.

Jean Leong