Team:Beijing Normal/Project


(Difference between revisions)
(The Experiments)
(The Experiments)
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*2 PCR
*2 PCR
**2.1 The pfu/Tag complex system
** 2.1 The pfu/Tag complex system
  Reagent           Concentration/Activity       50ul in total
  Reagent           Concentration/Activity       50ul in total
   taq buffer        10x                         5       
   taq buffer        10x                         5       
Line 58: Line 58:
   Template DNA         changeable                 0.5    
   Template DNA         changeable                 0.5    
   ddH2O             add to 50ul                 41
   ddH2O             add to 50ul                 41
**2.2 The program under pfu/Tag complex system
** 2.2 The program under pfu/Tag complex system
Progress     Program I                  
Progress     Program I                  
   Predenaturing    95℃        5 min          
   Predenaturing    95℃        5 min          

Revision as of 09:50, 12 August 2008

thum Home The Team The Project Parts Submitted to the Registry Modeling Notebook



We are aiming to create some magic intelligent bacteria to track and ‘eat’ pollutants PCBs (Polychlorinated Biphenyl) and dioxins efficiently, based on the methods of synthetic biology.

Polychlorinated biphenyls (PCBs) are a family of compounds produced commercially by the direct chlorination of biphenyl using ferric chloride and/or iodine as the catalyst.The biphenyl molecule is made up of two connected rings of six carbon atoms each, and a PCB is any molecule having multiple chlorines attached to the biphenyl nucleus.

Two distinct classes of bacteria have now been identified that biodegrade PCBs by different mechanisms, including aerobic bacteria which live in oxygenated environments and anaerobic bacteria which live in oxygen free environments such as aquatic sediments. The aerobes attack PCBs oxidatively , breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.

Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) were introduced into the biosphere on a large scale as by-products from the manufacture of chlorinated phenols and the incineration of wastes. Due to their high toxicity they have been the subject of great public and scientific scrutiny.

The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle. Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia.However, higher chlorinated dioxins requires anaerobic degradation process.

Organic pollutants such as PCB and dioxins, produced in human beings activities in the last century, are toxic and carcinogenic which are able to promulgate widely and accumulate to a high level of concentration by food chain. Due to their inherent thermal and chemical stability, it is commonly considered as indestructible under normal incineration or burial.

Nonetheless, by endowing some bacteria ability of utilizing such molecules as carbon source, cooperative evolution makes all possible! Enzymes assembled from related degradation pathways into our host strain serve as the function part. We introduce popular components involved in chemotaxis, quorum-sensing to regulatory parts, sense the environment signal, respond to move, accelerate growing and produce related degradation enzymes. After the cleaning work being finished, bacteria will return to the normal state.

Taking the condition of our lab into account, we decide just deal with the aerobic degradation path way.

to be continued and refreshed...

Project Details

Part 2

The Experiments

  • 1.Get the parts from biobrick

We largely follow the instructions provided by the webpage, however, more TE buffer is added(10ul). It seems that this will increase the amount of the plasmids dissolved and improve transformation effeciency.

  • 2 PCR
    • 2.1 The pfu/Tag complex system
Reagent	           Concentration/Activity	       50ul in total	
  taq buffer     	    10x	                         5      
  pfu/taq complex		                        0.8~1.0	
  dNTPmix	            10mM each	                 4	  
  Primer 1 	            10uM	                 1.5	  
  Primer 2	            10um	                 1.5	  
  Template DNA	         changeable	                 0.5	  
  ddH2O	             add to 50ul	                 41
    • 2.2 The program under pfu/Tag complex system

Progress Program I

  Predenaturing     95℃        5 min	         
  Denaturing	    95℃        30sec	 
  Annealing	    (Tm-5) ℃   30sec	         
  Extension	    72℃        theoretically 1min/1kb
  Last extention    72℃       5min
  Hold               4℃
  • 3 restriction enzyme digestion

select suitable enzymes and buffer. Analyse the system using NEB cutter in case of double digestion. NEB cutter finder. 37℃ water bath for 2~3h, 4h is preferable

  • 4 ligation

The key to a successful ligation according to our experience is avoiding high temperature. After a gel extraction with 32ul elution buffer, a approximately 20ul product is obtained. Then mixed with 20ul Buffer 1(Takara Ligation Kit). Reaction at 16℃ water bath is widely recommended, and time for ligation we use is 2h or more.

  • 5 Transformation


Part 3