Difference between revisions of "Part:BBa K759001:Experience"

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Escherichia coli bacterial culture containing plasmids BBa_K759001 (coding region of Ag43 only), was inoculated overnight in LB-chloramphenicol medium in a shaking water bath at 370C. Plasmid mini preps were conducted with a Qiagen plasmid mini prep kit. Single and double restriction digests were carried out accordingly:
 
Escherichia coli bacterial culture containing plasmids BBa_K759001 (coding region of Ag43 only), was inoculated overnight in LB-chloramphenicol medium in a shaking water bath at 370C. Plasmid mini preps were conducted with a Qiagen plasmid mini prep kit. Single and double restriction digests were carried out accordingly:
  
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[[File:Restriction_digest_table.png|Restriction_digest_table.png]]
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<center>[[File:Restriction_digest_table.png|.png]]</center>
  
 
Restriction digests with EcoRI, XbaI, SpeI and PstI was performed to linearize the vector and to confirm the existence of unique restriction sites, as required by the iGEM biobrick Assembly standard 10 rules. Double restriction digests with EcoRI plus PstI and XbaI plus SpeI were performed to separate the BBa_K759001 insert from the pSB1C3 plasmid backbone. Digests were incubated at 370C for 2 hours and analysed by gel electrophoresis. Theoretical maps were used to calculate the expected sizes of the fragments and to compare them against the gel electrophoresis results.  
 
Restriction digests with EcoRI, XbaI, SpeI and PstI was performed to linearize the vector and to confirm the existence of unique restriction sites, as required by the iGEM biobrick Assembly standard 10 rules. Double restriction digests with EcoRI plus PstI and XbaI plus SpeI were performed to separate the BBa_K759001 insert from the pSB1C3 plasmid backbone. Digests were incubated at 370C for 2 hours and analysed by gel electrophoresis. Theoretical maps were used to calculate the expected sizes of the fragments and to compare them against the gel electrophoresis results.  
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The plasmid was sequenced by the DNA Sequencing Services at University of Dundee .Sequencing analysis was performed on BBa_K759001 to confirm the suspected additional PstI sites. 6 sequencing primers (see Fig.1) were designed that were located evenly across BBa_K759001 (sequence data was taken from the Registry of Standard Biological Parts) plus both BBa_G00100 (forward primer that amplifies from the prefix) and BBa_G00101 (reverse primer that amplifies from the suffix), as defined by iGEM, were used to sequence Ag43 flanked by the prefix and the suffix.
 
The plasmid was sequenced by the DNA Sequencing Services at University of Dundee .Sequencing analysis was performed on BBa_K759001 to confirm the suspected additional PstI sites. 6 sequencing primers (see Fig.1) were designed that were located evenly across BBa_K759001 (sequence data was taken from the Registry of Standard Biological Parts) plus both BBa_G00100 (forward primer that amplifies from the prefix) and BBa_G00101 (reverse primer that amplifies from the suffix), as defined by iGEM, were used to sequence Ag43 flanked by the prefix and the suffix.
  
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[[File:Sequencing_primers.png|Sequencing_primers.png]]
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<center>[[File:Sequencing_primers.png]]</center>
  
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'''• Results'''
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'''• Results:'''
  
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[[File:restriction_digest_on_BBa_K759001.png|Sequencing_primers.png]]
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[[File:restriction_digest_on_BBa_K759001.png]]
  
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                Figure 1.  Gel electrophoresis on standard restriction digests of BBa_K759001
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Figure 1.  Gel electrophoresis on standard restriction digests of BBa_K759001
 
Restriction digests were separated on a 0.8% agarose gel. Lane 1; NEB 1 kb size standards ladder, Lane 2; uncut plasmid, Lanes 3-6; plasmid cut with either EcoRI, XbaI, SpeI or PstI respectively, Lane 7; plasmid cut with EcoRI+PstI, Lane 8; plasmid cut with XbaI+SpeI.
 
Restriction digests were separated on a 0.8% agarose gel. Lane 1; NEB 1 kb size standards ladder, Lane 2; uncut plasmid, Lanes 3-6; plasmid cut with either EcoRI, XbaI, SpeI or PstI respectively, Lane 7; plasmid cut with EcoRI+PstI, Lane 8; plasmid cut with XbaI+SpeI.
  
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[[File:Fragment sizes_BBa_K759001.png|Sequencing_primers.png]]
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<center>[[File:Fragment sizes_BBa_K759001.png]]</center>
 
              
 
              
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                      Figure 4. Fragment sizes resulted from standard restriction digests on BBa_K759001.
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<center>Figure 4. Fragment sizes resulted from standard restriction digests on BBa_K759001</center>
  
  
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[[File:Assembly sequence_BBa_K759001.png|Sequencing_primers.png]]
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<center>[[File:Assembly sequence_BBa_K759001.png]]</center>
  
 
Based on the sequencining data, a PstI restriction map of the pSB1C3-BBa_K759001 construct was  drawn using SnapGene Viewer:
 
Based on the sequencining data, a PstI restriction map of the pSB1C3-BBa_K759001 construct was  drawn using SnapGene Viewer:
  
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[[File:Plasmid_map_BBak759001.png|Sequencing_primers.png]]
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<center>[[File:Plasmid_map_BBak759001.png|450px|]]</center>

Revision as of 23:08, 3 October 2014


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K759001

User Reviews

UNIQd57af7b621a92bbd-partinfo-00000000-QINU UNIQd57af7b621a92bbd-partinfo-00000001-QINU

• Aim – to verify Assembly Standard 10 compliance


• Methods:


1. Standard restriction digest and gel electrophoresis

Escherichia coli bacterial culture containing plasmids BBa_K759001 (coding region of Ag43 only), was inoculated overnight in LB-chloramphenicol medium in a shaking water bath at 370C. Plasmid mini preps were conducted with a Qiagen plasmid mini prep kit. Single and double restriction digests were carried out accordingly:

.png

Restriction digests with EcoRI, XbaI, SpeI and PstI was performed to linearize the vector and to confirm the existence of unique restriction sites, as required by the iGEM biobrick Assembly standard 10 rules. Double restriction digests with EcoRI plus PstI and XbaI plus SpeI were performed to separate the BBa_K759001 insert from the pSB1C3 plasmid backbone. Digests were incubated at 370C for 2 hours and analysed by gel electrophoresis. Theoretical maps were used to calculate the expected sizes of the fragments and to compare them against the gel electrophoresis results.

2. Sequencing data

The plasmid was sequenced by the DNA Sequencing Services at University of Dundee .Sequencing analysis was performed on BBa_K759001 to confirm the suspected additional PstI sites. 6 sequencing primers (see Fig.1) were designed that were located evenly across BBa_K759001 (sequence data was taken from the Registry of Standard Biological Parts) plus both BBa_G00100 (forward primer that amplifies from the prefix) and BBa_G00101 (reverse primer that amplifies from the suffix), as defined by iGEM, were used to sequence Ag43 flanked by the prefix and the suffix.

Sequencing primers.png

• Results:

Restriction digest on BBa K759001.png

Figure 1. Gel electrophoresis on standard restriction digests of BBa_K759001 Restriction digests were separated on a 0.8% agarose gel. Lane 1; NEB 1 kb size standards ladder, Lane 2; uncut plasmid, Lanes 3-6; plasmid cut with either EcoRI, XbaI, SpeI or PstI respectively, Lane 7; plasmid cut with EcoRI+PstI, Lane 8; plasmid cut with XbaI+SpeI.

Fragment sizes BBa K759001.png
Figure 4. Fragment sizes resulted from standard restriction digests on BBa_K759001


Assembly of the sequencing data was performed manually using the CLUSTAL OMEGA multiple alignment program. The resulting sequence (query) was then compared against the original sequence for BBa_K759001 (subject) (sequence of 4493 bp, as taken from the Registry of Standard Biological Parts), using BLAST Pairwise sequence alignment viewer:


Assembly sequence BBa K759001.png

Based on the sequencining data, a PstI restriction map of the pSB1C3-BBa_K759001 construct was drawn using SnapGene Viewer:

Plasmid map BBak759001.png