Saturday, August 16, 2008

Week 8 SIP - Chloroform Shock

Hello everyone. It is the 8th week of SIP/MP and its my turn to blog again. This week I will blog on Chloroform Shock its relevance to my project.

Chloroform Shock is a method which is commonly used in the extraction of periplasmic proteins of gram-negative bacteria. Gram-negative bacteria is not only enclosed by its cell membrane, but also a periplasm. Many periplasmic proteins can be found within the periplasmic space (the region between the inner and outer membrane) of the bacteria. Hence, to extract these periplasmic proteins, chloroform is used.

Chloroform is a solvent which is relatively unreactive, miscible and volatile. It has to be said the exact mechanism of action of chloroform shock is currently not known. However, scientiests have been using this technique to extract periplasmic proteins. It is postulated that chloroform form pores in the outer cell wall of the gram-negative bacteria and releasing periplasmic proteins out into the extracellular environment. The optimal time for chloroform to work is 15 minutes for extract of periplasmic proteins only. If the time taken is too short, it may not have reached/penetrate the periplasmic space and periplasmic proteins are not extracted. When the time taken is too long, it may penetrate the periplasmic space and will extract other intracellular proteins as well, such as cell membrane proteins which is irrelevant to my project. Thus, great care must be taken to ensure reaction of the bacteria (cell pellet) with chloroform is 15 minutes exactly.

The use of chloroform is not without its risks. The bottle of chloroform is always opened within the Biosafety Level II Cabinet (which i am using) and not outside it. This is because accidental inhalation can be bad for the health. The role of the Biosafety Cabinet level II is to protect the user as well as the speciman. In Class II cabinets, there is always stream of inward air moving into the cabinet. This is called the inflow and prevents the aerosol generated during any microbial work, to escape out of the Cabinet (air can only flow into the BSC but not out). The inflow can only reach the front inlet grill, just in front of the operator. This is to ensure that unfiltered air outside the BSC cannot enter the BSC and thus there will not be any contamination. A special feature of BSC Level II cabinet is the HEPA-filtered air stream which causes air stream to flow downwards inside the BSC after sucking air from above and filtered. This flushing is called downflow and protects samples within BSC from contamination. In the case where chloroform is accidentally inhaled or consumed, it can depress the nervous system and cause dizziness, fatigue and headache. Hence the appropriate personal protective equipment to wear is a pair of gloves, labcoat and work inside the Biosafety Level II cabinet when performing chloroform shock.

Basic Outline of Chloroform Shock (sorry i cannot give the full details such as centrifuge speed, volume, etc as my supervisor do not allow, hence here is only the basic workflow)

1. Centrifuge to obtain cell pellet (from broth culture)
2. Wash cell pellet
3. Resuspend with PBS (phosphate buffer saline)
4. Repeat step 1 and 2 three times
5. Take OD600 reading
6. Calculate volume of cells to dispense into 5 eppendorf tubes
7. Centrifuge the 5 eppendorf tube
8. Decant supernatant and mix gently
9. Add chloroform and incubate 15 minutes
10. Add Tris-HCl
11. Centrifuge the eppendorf tubes
12. Extract the periplasmic proteins from the supernatant

Thanks for taking your time to read my entry and have an enjoyable and fruitful SIP for the next 12 weeks.

From: Ma Xianwei Benjamin
Class: TG01
0606181F

21 comments:

group1 said...

Hello Ben :)

Why must it be 5 eppendorf tubes?

Yvonne.

tg01 group 2 said...

Hello yvonne,

After taking of OD600, the calculated volume of cells is transfered into 5 eppendorf tubes. This is to allow the cell pellet to be more concentrated. And subsequently the addition of chloroform to 5 tubes is better than 1 as there is a larger syrface area for the reaction to take place, whereas in 1 tube where all the volume of cells is pooled together the reaction with chloroform is slower.

Thanks

From: Benjamin
class:tg01

tg01 group 2 said...

Hi Benjamin

Just to let you know. Gram positive also has periplasmic space. This space is between peptidoglycan and plasma membrane. Some people say that gram positive does not have periplasm is because it is very very small compared to gram neg.

Since it is beside the peptidoglycan, it is involve in the synthesis of the peptidoglycan wall. Electron transport chain also may occur there.

My question is
Are there any other methods besides chloroform shock? Is it similar to osmotic shocks like sucrose shock, NaCl shock?

Thank you
Ernest TG01
0606330i

tg01 group 2 said...
This comment has been removed by the author.
tg01 group 2 said...

Hello Ernest,

yes you are right that gram-positive have periplasm but is very much smaller than gram-negative bacteria. However in my project application I will be using chloroform shock to extract periplasmic proteins fromgram-negative bacteria (S.maltophilia). I did not say that gram-positive bacteria do not have periplasmic space so please don't get the wrong idea.

Two other methods used to extract periplasmic proteins are lysozyme digestion and osmotic shock. For further details please refer to this website: http://www.epibio.com/item.asp?ID=327 as it is much more comprhensive.

From: Ben

THE CODEC 5 said...

Hi Ben,

What is the function of adding tris-HCl after the 15 min incubation?

Thanks
Xin Yi
TG02

~immortals~ said...

Hi Ben!!

Periplasmic proteins are made up of what? What are the functions of periplasmic space and proteins for the gram-negative bacteria?

What happens if you accidentally spill some chloroform in the BSC?

Thanks!!
Amir

tg01 group 2 said...

Hello Xinni,

Good question... the function of adding Tris-HCl is to dissolve or solublise the periplasmic proteins after they have been extracted by the chloroform (after 15 minutes incubation). This is to ensure that after centrifugation, the periplasmic proteins(found in the supernatant)can be seperated from the chloroform.

Thanks!

From: Ben

tg01 group 2 said...

Hello Amir

As periplasmic proteins are proteins, they are made up of amino acids. As currently the identity of the proteins are not know and i have to find out in my project, I am unable to tell you the exact name of the periplasmic protein.

As Ernest mentioned, some of the functions of periplasmic space include the synthesis of peptidoglycan wall and electron transport chain. It can also acts as virulence factors of some bacterias by possessing some eznymes to degrade antibiotics so they become antibiotic resistant.

If chloroform is spilled in the BSC, just spray with 70% ethanol and wipe the area with a paper towel. Usually i will not spill it and be extra careful as it is the only bottle of chloroform in the lab. If i spill it, i cant do my experiments :)

Thanks!

From: Ben

MadTechs said...

Hey..

you mentioned about "HEPA-filtered air stream".. what does HEPA actually stands for?

thanks C:

Nur Azeimah
0607060A
TG02

THE CODEC 5 said...

Hi Ben,

Just to tell you, I am xin yi and not xinni. so you should be replying me, xin yi.

Thanks
Xin Yi
TG02

tg01 group 2 said...

Hello Xin Yi,

Lol... sorry for my mistake (very blur that day) and thanks for taking ur time go through my entry. Tis-HCl is as solvent to dissolve the periplasmic proteins so that it can be seperated from the cell pellet and chloroform after centrifugation.

From: Ben

tg01 group 2 said...

Hello Nur Azeimah,

HEPA stands for high efficiecny particulate air. HEPA filter is a high efficiency filter that removes 99.97% or dust or air particles that are 0.3 microns in size. This helps to ensure the hood is free of dust and airborne contaminants.

Thanks!

From: Ben

group1 said...

Hi ben!

you know? i've use before chloroform to join acrylic plastic when i was taking DnT during sec4. i wonder if it's the same that you use in your lab.

ps. we didnt use biosafety cabinet back then. =X

thanks,
Yumei.

tg01 group 2 said...

Hello Yu mei,

Sorry i am not sure whether the chloroform i am using is the same as that used in the dnt lab. The chloroform i am using is transclucent (like water) and miscibile.

Thanks

imglad said...

hey ben

whats OD600?

and you mention that you want to take out this periplasmic proteins from the periplasmic space. so whats so special about these periplasmic proteins? are they like special proteins that only exist in the periplasmic space? how different are they from the intracellular proteins?

thanks
GLAD

tg01 group 2 said...

Hello Gladys,

The purpose of taking OD600 is to calculate the number of bacterial cells present in the broth culture. Optical density is measured using a cell density meter. Is function is to measure the concentration of bacteria in the culture. The machine passes a visible light through a cell suspension/culture and light is scattered. Greater scatter is indicated by high OD600.The growth of bacteria is high or other materials/contaminants such as dust is present when OD600 is high.
OD600 is low when there is little growth of bacteria.

The periplasmic proteins of bacteria are known to be virulent in many bacteria genus and species. Hence, it is suspected that S.maltophilia's periplasmic proteins may play a role in its pathogenicity. As there is little research done on this bacterium, there is little information on what these periplasmic proteins can do/pathogenic. Hence it is important to first identify the periplasmic protein before comparing it to known bacteria species such as E.Coli to have a better idea of the pathogenicity of S.maltophilia periplasmic proteins. One function of periplasmic proteins is that it can breakdown antibiotics hence it is very antibiotic resistant and only vancomycin can treat it.

Proteins are made in the cytoplasm and transported into the various parts of the cell/bacteria. THose proteins transported to the periplasm are termed as periplasmic proteins. These are proteins that are found in the periplasmic space which is in between the inner and outer membrane of the bacteria. Those proteins that are produced in the cytoplasm but do not get transported out of the cell(secretory proteins) or stay in the periplasm (periplasmic proteins) stay within the cell and play important roles in the cell such as cell-cell signaling mechanism, generate ATP for cellular survival and transport functions (transport vesicles).

Some Periplasmic proteins can exhibit enzymatic activity. These enzymatic activity are produced by periplasmic proteases. It has been found out that these periplasmic proteases show higher activity at 37 degree celsius as compared to 28degree celsius.Hence it can be infered that the bacterium may be clinically significant at 37 degree celsius (temperature within the host). So the main objective is to identify this periplasmic protease which is a periplasmic protein and establish its role as a virulent factor in S.maltophilia.

Thanks and I hope you understand more after the explanation!!

From: Benjamin Ma
Class: TG01
0606181F

imglad said...

noted ben.

thanks(:
GLAD

De Incredibles said...

Hey ben,

just wanna know, what are the uses of periplasmic proteins and what are you gonna use them for in your project. Thanks.

Debbie
TG02

tg01 group 2 said...

Hi Debbie,

Periplasmic proteins are virulence factors of S.maltophilia and im going to study by finding out the identity (name) of the periplasmic proteins in my project.

Thanks!

From: Benjamin Ma
Class: TG)1
0606181F

Ms_chew said...

Keep up the good job.