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Team:Beijing Normal/Project
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Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more. | Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more. | ||
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Revision as of 11:55, 7 September 2008
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Overall
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 ctalyst.The total amount of PCBs produced in the world is estimated 1.2 million tons.Because PCBs have been released into the environment in many countries over decades, these compounds have become serious and global environmental contaminants.PCBs tend to accumulate in biota owing to their lypophilic property.
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. And do some work on increasing the degradation effeciency.
to be continued and refreshed...
Project Details
background information
- Although many environmental pollutants are efficiently degraded by microorganisms, others such as PCBs and dioxins persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.
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/Taq 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 -- ddH2O --- add to 50ul
- 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. [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp 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
Add 10ul ligation product into 100ul competence cells(Top10 or others) and keep it in 4℃ refrigeratory for 30min. After a hotshock of 45sec(for chemical competent) or 90sec(for CaCl2 competent), the cells are placed in the 4℃ refrigeratory again for 3-5min. Then add 600~800ul SOC to hotshock cells and incubate in 37℃ shaking table for 1h(160rpm-180rpm). At last the cells are palced on petri dishes with relative antibiotics at appropriate concentration. Then place in 37℃(temperature accords to the properties of different hosts and plasmids),incubate for 12h or more.
