Difference between revisions of "Team:Linkoping Sweden/Results"

Revision as of 13:57, 13 October 2016

Result

The original plan for Liu iGEM was to utilize the standard assembly method to establish the final construct. However, the effectivity of this method was quite low and the approach on the assembly needed to be modified. The Gibson Assembly method was therefore selected, with its main advantage to assemble all DNA fragments in one step. By utilizing Gibson Assembly the transformation was successful and plenty of colonies were obtained. Although, not all of them contained the complete construct with all the DNA fragments of interest. The colonies with the proper base length were sequenced and it could be determined that Cas9 and LIP had successfully been inserted into the vector, whilst sgRNA, U6 and Hygromycin cassette (Hyg) were not incorporated. Fortunately, a yellow fluorescent protein (YFP) was inserted into the construct to substitute for the Hyg selection. Furthermore, three constructs with the fragments LIP, LIP-RFP and UTR were successfully obtained.

A big part of Liu iGEM’s project is the alga Chlamydomonas reinhardtii. The algae successfully grew both in light and dark conditions. The constructed vector, which included YFP instead of Hyg, was transformed into the algae with electroporation. Since no hygromycin resistant could be utilized everything grew on the plates and there was no selection on the plates. The colonies were screened with fluorescent microscope, but because of the difficulties to detect YFP the conclusion was that the electroporation was not successful.

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 ==Result==
-The original plan for Liu iGEM’s project was to utilize the E1010 biobrick as the main vector. E1010 is in itself a standard pSB1C3 vector with a RFP gene. The idea was to delete this gene by utilizing restriction enzymes, and instead insert a DNA fragment of interest. For this method, each DNA fragment of interest requires a digestion before the fragment can be assembled into the pSB1C3 vector. When performing this method, the transformation was repeatedly unsuccessful and because of this, iGEM’s standard linear pSB1c3 vector was instead selected. Despite utilizing iGEM’s pSB1c3 linear vector the ligation of the DNA fragments were ineffective. The reason being that the backbone did not consist of the right restriction sites which could be analyzed via SnapGene software.
+The original plan for Liu iGEM was to utilize the standard assembly method to establish the final construct. However, the effectivity of this method was quite low and the approach on the assembly needed to be modified. The Gibson Assembly method was therefore selected, with its main advantage to assemble all DNA fragments in one step. By utilizing Gibson Assembly the transformation was successful and plenty of colonies were obtained. Although, not all of them contained the complete construct with all the DNA fragments of interest. The colonies with the proper base length were sequenced and it could be determined that Cas9 and LIP had successfully been inserted into the vector, whilst sgRNA, U6 and Hygromycin cassette (Hyg) were not incorporated. Fortunately, a yellow fluorescent protein (YFP) was inserted into the construct to substitute for the Hyg selection. Furthermore, three constructs with the fragments LIP, LIP-RFP and UTR were successfully obtained.
-To complete the project in time the approach on the assembly needed to be modified. The Gibson Assembly method was therefore selected, with its main advantage to assemble all DNA fragments in one step. By utilizing Gibson Assembly the transformation was successful and plenty of colonies were obtained. Although, not all of them contained the complete construct with all the DNA fragments of interest. The colonies with the proper base length were sequenced and it could be determined that Cas9 and LIP had successfully been inserted into the vector, whilst sgRNA, U6 and Hyg were not incorporated. Fortunately, an YFP were inserted into the construct to substitute for the Hyg selection. Furthermore, three constructs with the fragments LIP, LIP-RFP and UTR were successfully obtained.
+A big part of Liu iGEM’s project is the alga Chlamydomonas reinhardtii. The algae successfully grew both in light and dark conditions. The constructed vector, which included YFP instead of Hyg, was transformed into the algae with electroporation. Since no hygromycin resistant could be utilized everything grew on the plates and there was no selection on the plates. The colonies were screened with fluorescent microscope, but because of the difficulties to detect YFP the conclusion was that the electroporation was not successful.
-A big part of Liu iGEM’s project is the alga Chlamydomonas reinhardtii. The algae successfully grew both in light and dark conditions. The constructed vector, which included YFP instead of Hyg, was transformed into the algae with electroporation. Since no hygromycin resistant could be utilized everything grew on the plates and there was no selection on the plates. The colonies were screened with fluorescent microscope, but because of the difficulties to detect YFP the conclusion was that the electroporation was not successful.
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