840:153g:Projects/project24

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 17: Line 17:
* Name of student Parul Sirohi
* Name of student Parul Sirohi
-
==Over expression of diacyl- glycerol acyl transferase (DGAT) in ''Chlamydomonas reinhardtii to enhance tri-acyl glycerol production, the precursor of Bio-fuel production.==
+
==Over expression of Acetyl- CoA carboxylase (ACC)sub-unit accC in E.coli to enhance fatty acid accumulation for Bio-fuel production”.==
-
* Gene used: Diacyl glycerol acyl transferase (DGTA) gene
+
* Target Organism: E. coli 0157:H7�
-
 
+
Source: Biology department of University of Northern Iowa�
-
Function of gene: DGTA is a precursor enzyme which regulates the final bio-synthetic step of Triacyl glycerol production ( fatty acid which is used to produce bio-fuel).
+
Media: Luria Broth�
 +
Gene: Acetyl CoA carboxylase biotin carboxylase (accC)
 +
Assembly #: NC_011353.1 Region: 4242644..4243993�
 +
Introns: none
 +
Bio-brick Compatibility: Compatible
 +
Plasmid used: Vector Plasmid pSB1A3
 +
Promoter used:Part: BBa_J23100
 +
PCR primers for accC gene:bp
 +
              1                                                        30                       
 +
Template  5’ A-T-G-C-T-G-G-A-T-A-A-A-A-T-T-G-T-T-A-T-T-G-C-C-A-A-C-C-G-C  3’
 +
FP:      5’  T-A-C-G-A-C-C-T-A-T-T-T-T-A-A-C-A-A-T-A-A-C-G-G-T-T-G-G-C-G  3’
 +
                        
 +
            1301                                                            1333                                     
 +
Template: 5’ G-C-A-C-T-A-A-C-A-T-C-C-A-C-T-A-T-C-T-G-G-A-G-A-A-A-A-A-A-C-T-C-G 3’
 +
RP:      5’ C-G-T-G-A-T-T-G-T-A-G-G-T-G-A-T-A-G-A-C-C-T-C-T-T-T-T-T-T-G-A-G-C 3’
-
Introduction:
 
-
Transgenic microalgae has been the promising organism to study for biofuel production. Due to its ability to grow fast and product lipid, microalgae is the choice of organism for us. Specially in microalgae we are interested to see if the over expression of DGTA gene will enhance Triacyl glycerol production (TAG). TAG is the final by-product of fatty acid synthesis in micro algae. And DGTA is the regulatory enzyme for TAG bio-synthesis. The global synthesis pathway of TAG synthesis in cells ic comprised of three major steps:
 
-
1. Carboxylation of acetyl- CoA to form malonyl CoA
+
 
-
2.Acyl chain elongation
+
'''Function of gene''': accC is a precursor enzyme which regulates the intermediate bio-synthetic step of Triacyl glycerol production ( fatty acid which is used to produce bio-fuel)
-
3.TAG formation.
+
 +
'''Introduction''':The gene of interest in this project is accC ( Acetyl Co-A carboxylase biotin carboxylase).This gene catalyze the formation of malonyl-CoA substrate for biosynthesis of fatty acid synthesis.
 +
ACC is a multi subunit( accA, accB, accC and accD) enzyme in most prokaryotes. It is also found in the chloroplast of most of plant and algae.
 +
Fatty acid is the major prerequisite for bio-fuel production, so its over production might enhance bio-fuel production.
 +
Overexpression of the enzyme DGAT  is most likely to enhance  lipid, Tri-acylglycerol over production but due to high introns numbers in the source ( Arabidopsis thaliana) we chose  ACC over DGAT
 +
'''3 major biochemical steps:'''
 +
1. Carboxylation of acetyl- CoA to form malonyl CoA by enzyme ACC which has four sub-unit (accA,accB,accC and accD)
 +
2.Acyl chain elongation
 +
3.TAG formation.
    
    
-
* List all the steps that are needed to complete your project
+
* [['''Steps for project:''']]
 +
1.Grow the source organism (E. coli)
 +
2.DNA extraction from the source (E. coli)
 +
3.Electrophoresis to check desired DNA segment (bp)
 +
4.Primer designing
 +
5.Multiplication of  gene of interest by PCR
 +
6.Electrophorosis
 +
7.Digestion of Plasmid by restriction enzymes ( cut plasmid with S+P i.e bp2 and gene of interest with X+P i.e bp 2149 and P at bp20 )
 +
8.Ligation of accC gene in plasmid vector (pSB1A3)
 +
9.Transformation of vector plasmid  into host organism E. coli
 +
10.Cloning of cells in a LB media
 +
11.Selection for recombinant DNA colonies by antibiotic selective media (LB+ ampicillin)
 +
12.Inoculation of E.coli in biomass
 +
13.Testing of fatty acid  by High pressure thin layer chromatography
* Do not go into experimental details and don't list procedures. Just list the major steps necessary 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
* Please also make yourself familiar with uploading pictures and *.ppt files
* Please also make yourself familiar with uploading pictures and *.ppt files

Revision as of 00:20, 6 September 2012

Search this Project

Customize your entry pages

Team Members

  • Name of student Sanju Timilsina
  • Name of student Parul Sirohi

Over expression of Acetyl- CoA carboxylase (ACC)sub-unit accC in E.coli to enhance fatty acid accumulation for Bio-fuel production”.

  • Target Organism: E. coli 0157:H7�

Source: Biology department of University of Northern Iowa� Media: Luria Broth� Gene: Acetyl CoA carboxylase biotin carboxylase (accC)� Assembly #: NC_011353.1 Region: 4242644..4243993� Introns: none Bio-brick Compatibility: Compatible Plasmid used: Vector Plasmid pSB1A3 Promoter used:Part: BBa_J23100 PCR primers for accC gene:bp

             1                                                        30                        

Template 5’ A-T-G-C-T-G-G-A-T-A-A-A-A-T-T-G-T-T-A-T-T-G-C-C-A-A-C-C-G-C 3’ FP: 5’ T-A-C-G-A-C-C-T-A-T-T-T-T-A-A-C-A-A-T-A-A-C-G-G-T-T-G-G-C-G 3’  

           1301                                                             1333                                      

Template: 5’ G-C-A-C-T-A-A-C-A-T-C-C-A-C-T-A-T-C-T-G-G-A-G-A-A-A-A-A-A-C-T-C-G 3’ RP: 5’ C-G-T-G-A-T-T-G-T-A-G-G-T-G-A-T-A-G-A-C-C-T-C-T-T-T-T-T-T-G-A-G-C 3’


Function of gene: accC is a precursor enzyme which regulates the intermediate bio-synthetic step of Triacyl glycerol production ( fatty acid which is used to produce bio-fuel)

Introduction:The gene of interest in this project is accC ( Acetyl Co-A carboxylase biotin carboxylase).This gene catalyze the formation of malonyl-CoA substrate for biosynthesis of fatty acid synthesis. ACC is a multi subunit( accA, accB, accC and accD) enzyme in most prokaryotes. It is also found in the chloroplast of most of plant and algae. Fatty acid is the major prerequisite for bio-fuel production, so its over production might enhance bio-fuel production. Overexpression of the enzyme DGAT is most likely to enhance lipid, Tri-acylglycerol over production but due to high introns numbers in the source ( Arabidopsis thaliana) we chose ACC over DGAT

3 major biochemical steps:

1. Carboxylation of acetyl- CoA to form malonyl CoA by enzyme ACC which has four sub-unit (accA,accB,accC and accD) 2.Acyl chain elongation 3.TAG formation.


  • [[Steps for project:]]

1.Grow the source organism (E. coli) 2.DNA extraction from the source (E. coli) 3.Electrophoresis to check desired DNA segment (bp) 4.Primer designing 5.Multiplication of gene of interest by PCR 6.Electrophorosis 7.Digestion of Plasmid by restriction enzymes ( cut plasmid with S+P i.e bp2 and gene of interest with X+P i.e bp 2149 and P at bp20 ) 8.Ligation of accC gene in plasmid vector (pSB1A3) 9.Transformation of vector plasmid into host organism E. coli 10.Cloning of cells in a LB media 11.Selection for recombinant DNA colonies by antibiotic selective media (LB+ ampicillin) 12.Inoculation of E.coli in biomass 13.Testing of fatty acid by High pressure thin layer chromatography

  • Do not go into experimental details and don't list procedures. Just list the major steps necessary to complete your project
  • Please also make yourself familiar with uploading pictures and *.ppt files

Important Results and Milestones

  • keep track of your most important results and refer to the corresponding page in your notebook
  • 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).

Recent changes




Personal tools