Part:BBa_K346007:Experience
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===Applications of BBa_K346007===
2014 iGEM Aberdeen team
• Aim – to verify Assembly Standard 10 compliance
• Method:
Standard restriction digest and gel electrophoresis
Escherichia coli bacterial culture containing plasmids BBa_K346007 (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:
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_K346007 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.
• Result:
Figure 1. Gel electrophoresis on standard restriction digests of BBa_K346007.
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.
Figure 2.Fragment sizes resulted from standard restriction digests on BBa_K346007.
• Conclusion:
The pSB1C3-BBa_K346007 construct was shown to contain 6 additional PstI sites, as found in the wild-type gene of Antigen 43. This part is the basic part of BBa_K759001 Biobrick, which we have also shown that contains the same PstI sites (see Experience section of BBa_K759001).
Thus we conclude that the BioBrick BBa_K346007 originally deposited with iGEM did not meet assembly standard 10, and the 6 native PstI sites present in Ag43 had not in fact been removed as stated in the original documentation.
Based on the results mentioned above, pSB1C3-BBa_K759001 has been chosen as the subject for removal of the PstI sites to ensure that an Ag43 BioBrick is created that meets Assembly Standard 10 requirements (see the RFC10 compatible version of this Biobrick created by 2014 iGEM Aberdeen team:BBa_K1352000)
Bilkent-UNAMBG 2019 iGEM Team
Antigen 43 (Ag43) is found in E. coli and functions as a autotransporters under the family of self associating autotransporters. Expressed Ag43 in E. coli result in the Ag43-Ag43 interactions which mediate the autoaggregation of the cell. Ag43 is cell surface exposed protein which can carry and display the cargo protein on the outer membrane. Autoaggregation of the cell is conferred by the a passenger domain. We aimed to improve this part by changing, deleting, the first 160 amino acids of the a passenger domain. With the deletion of first 160 amino acids which is responsible for the cell aggregation mediation, Ag43 structure become truncated and does not confer the cell to cell aggregation. Also, we have added one more stop codon at the end of the Ag43 by PCR. Our part's name is BBa_K3003028 which is the improvement of this part.
Materials and Methods :
By using primers specifically designed for amplifying the region that excludes first 160 amino acids of the alpha subunit. We have obtained the Ag43 from the E. coli genome by Colony PCR. Gene size was demonstrated in the agarose gel image which was prepared in concentration of %1.
Result: Ag43 containing plasmid was induced with the aTc after OD has reached to 0.4. Cells were monitored for 4 hours in a every hour period. At the end of 4 hours, we demonstrated that 160N deletion in alpha subunit did not confer to any cell to cell aggregation, on the other hand native Ag43 has depicted cell to cell aggregation.
Figure 1: Native Ag43 alpha subunit (Uninduced-Induced)
Figure 2: 160N deletion in alpha subunit confers to no aggregation (Uninduced-Induced)
Figure 3: Aggregation curve of native Ag43 and improved Ag43
User Reviews
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Kenta |
Aggregation was confirmed in the construct of BBa_K759001. But this part contains 6 PstI recognition sites in the 883-888, 1441-1446, 2041-2046, 2665-2670, 2936-2941, 2989-2994 bp regions at the time of 2012. |
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