Difference between revisions of "Part:BBa K2148001:Experience"

 
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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how you used this part and how it worked out.
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The aadA gene was assembled into a level 1 alpha plasmid with two other parts from our library: homology region with psaA 5' UTR and promoter and the 3' UTR rbcL with homology region. This generated a plasmid which could be used to transform <i> Chlamydomonas reinhardtii</i> chloroplasts by insertion through homologous recombination. We shot this construct into cultures of the CW15 <i>C. reinhardtii</i> biolistically.
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The cultures grew (as shown below in image) on spectinomycin+TAP plates, showing the functionality of this part. Colonies were indeed seen to have grown on the negative control plates but we believe this to be due to our own contamination in the procedure because:
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(a) This antibiotic resistance is not commonly used in the department.
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(b) Could possible be the case that a layer of cells has died (due to lack of resistance) and the new colonies are forming upon these, avoiding contact with the antibiotic. However, we replated the colonies onto new antibiotic+TAP plates and the cells were able to grow well.       
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PCR analysis was not conclusive on the result of the biolistic transformation. Ideally, would need to repeat the experiment (time constraints did not allow us to).
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<b> INCLUDE GEOFF'S PHOTOS OF COLONY </b>   
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===Applications of BBa_K2148001===
 
===Applications of BBa_K2148001===

Latest revision as of 19:03, 13 October 2016


The aadA gene was assembled into a level 1 alpha plasmid with two other parts from our library: homology region with psaA 5' UTR and promoter and the 3' UTR rbcL with homology region. This generated a plasmid which could be used to transform Chlamydomonas reinhardtii chloroplasts by insertion through homologous recombination. We shot this construct into cultures of the CW15 C. reinhardtii biolistically.

The cultures grew (as shown below in image) on spectinomycin+TAP plates, showing the functionality of this part. Colonies were indeed seen to have grown on the negative control plates but we believe this to be due to our own contamination in the procedure because:

(a) This antibiotic resistance is not commonly used in the department.

(b) Could possible be the case that a layer of cells has died (due to lack of resistance) and the new colonies are forming upon these, avoiding contact with the antibiotic. However, we replated the colonies onto new antibiotic+TAP plates and the cells were able to grow well.

PCR analysis was not conclusive on the result of the biolistic transformation. Ideally, would need to repeat the experiment (time constraints did not allow us to).

INCLUDE GEOFF'S PHOTOS OF COLONY


Applications of BBa_K2148001

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