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Cloning the gene of calcoaceticus strain ADP1 to observe its expression in E.coli.

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Team Members

  • Sanjana Gurrala
  • Sahil Sharma
  • Sindhu Adhikari

Cloning the gene of Acinetobacter calcoaceticus strain ADP1 to observe its expression in E.coli.

  • The sp.ADP1 is responsible for biodegradation of harmful chemicals. The catABCDE genes are responsible for conversion of catechol to intermediates of kreb's cycle. The first gene which helps catechol to catalse to catechol 1,2-dioxygenase is gene and the other cat genes catBCDE convert catechol 1,2-dioxygenase to succinate and acetyl co-A which are the intermediates of kreb's cycle. The genomic sequence of was taken from , it contains 936bp and its accession number is NC_005966.1. The gene will be amplified using PCR.The designed primers of catA gene are 18-GAS-F and 18-GAS-R and the of interest are Bba_I765007,Bba_K116401, Bba_K118011. Then, restriction enzymes are used to insert the cloned DNA into T-vector and the plasmid vector is transformed in E.coli and are grown in a growth medium under suitable conditions. After their growth, the plasmid is taken to check wethere the gene of interest is expressed.
  • List all the steps that are needed to complete your project
  • Do not go into experimental details and don't list procedures. Just list the major steps necessary to complete your project
  • http://www.openwetware.org/wiki/Image:Cloning_the_catA_gene_of_Acinetobacter_sp-2.ppt *.ppt files

Primers

Forward primer

18-GAS-F

5' atggaagtta aaatattcaa tactcagg 3'

Reverse primer

18-GAS-R

5' ttacaccgctagacgtgg 3'

Promoters

Bba_I765007- (Fe and UV promoter)

Bba_K116401-( external phosphate sensing promoter)

Bba_K118011-(Glucose repressible promoter)

Important Results and Milestones

Tuesday September 20, 2011 We prepared LB media and autoclaved it. While waiting for the media to autoclave we extracted our promoter part(Bba_K118011)from Biobrick Spring 2011 Kit 2 and stored it in tube at –80 degree for using it in next lab day.As the color of sample was red we were able to figure out that we successfully extracted the part from the kit.After autoclave was done we added Ampicillin to the media poured it into plates and stored the plate's for further use.

By Sindhu Adhikari.

Thursday September 22, 2011. Transformation of the Plasmid DNA of our promoter of choice BBa_K118011 and PsB1A7 DNA given by Axel was done. The required materials for Transformation were Competent cells, Plasmid DNA, SOC media, LB media plates. Firstly, Competent cells and Plasmid DNA was thawed on ice. Then, five tubes were taken. One of the tube had control which is competent cells and other were DNA samples of BBa_K118011 and PsB1A7 of different concentration. To these samples 50ul of competent cells were added and thawed on ice for 20 mins. In the meanwhile water bath was set to 42 C. After thawing on ice, samples were given heat shock for 50 secs at 42C then kept on ice for 2 mins. 1 ml of SOC media was added to all the tubes and kept in incubator shaker for an hour at 37C to increase the transformation efficiency of the Plasmid. After an hour the tubes were taken from the incubator shaker, 5 were plated with 200ul of sample in ampicillin plates and as control for our transformation two other plates were taken and 50ul of competent cells were added one with ampicillin and one without ampicillin to check the growth of competent cells. After transformation was done, plates were kept in incubator at 37c, overnight for bacteria to grow.

By Sanjana Gurrala.

Tuesday September 27,2011 The transformed plates were observed today.The growth of competent cells were observed in antibiotic media and antibiotic free media.So we are not sure about wether the ampicillin is not working or the competent cells have developed resistance for the ampicillin. Today we took 5 plates:- a) Lb media plate without antibiotic b) kanamycin plate and other other three were ampicillin plates which were prepared at different dates. All the plates were inoculated with 20ml of competent cells,to check wether the cells have developed the resistance for antibiotics.Then the plates were kept in the incubator for over-night and results will be obeserved tomorrow.

By:Sahil Sharma

  • upload important pictures (don't forget to label them! Powerpoint is very convenient). Remember: these will become quite handy later in your summary report or final presentation. If you do label and upload the pictures as soon as you got them, your summary report can be written much more effortlessly (do you usually procrastinate? This is chance to do some work before hand that frees you up for finals week).

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