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Applications of BBa_K542009

Datasheet for BBa_K542009 in E. coli strain BL21 (DE3).



The Peking 2010 iGEM team characterized Antigen 43 (AG43) using a T7 promoter expression system in E. coli BL21(DE3) strain and submitted the coding sequence with no promoter or ribosome binding site (RBS) to the parts registry (BBa_K364007). In order for AG43 to be used, BBa_K542009 was assembled to include the required regulatory elements. The pLacI promoter and RBS were added to the coding sequence for AG43.

Construct

Part Number: BBa_K542009

Figure 1. BBa_K542009. The inductive aggregation construct was made by assembling BBa_J04500 and BBa_K346007.

Sedimentation by Antigen 43

E. coli DH5α cells expressing BBa_K542009 were monitored for sedimentation and compared to sedimentation of uninduced cells.

Materials and Methods

Overnight cultures E. coli DH5α containing BBa_K542009 or BBa_J04500 (blank control) were grown with the appropriate antibiotics. Three 50 mL LB cultures – BBa_J04500 (control), BBa_K542009 (induced), and BBa_K542009 (uninduced) – with the appropriate antibiotics were inoculated to an OD₆₀₀ of 0.1 and incubated at 37°C with shaking.

At OD₆₀₀ of 0.4-0.6, the cultures were induced (excluding one of the BBa_K542009 cultures) with IPTG to a concentration of 0.001% and growth was monitored to a final OD₆₀₀ of 1. The cells were centrifuged at 5000 x g for 5 min and the supernatant discarded. The cells were resuspended with 5 mL of 1% PBS (with 0.15 mM NaCl) and transferred into test tubes. The cells in the test tubes were agitated and 100 µL was taken ~0.5 cm from the surface every 10 min for the first 60 min; then every hour for the next 3 hours and again at 18 hours and 18.5 hours. The samples were also subjected to OD₅₉₀ measurements using a microplate and the SoftMax Pro Software.

Results

Figure 2. E. coli DH5a cells containing BBa_K542009. Cultures were first agitated and then sedimentation rates were monitored. (a) Initial reading at the beginning of the experiment for the induced and uninduced. (b) Initial reading for the blank and 30 min after agitation for the induced and uninduced. (c) 3.5 hr after agitation for the blank and 4 hr after agitation for the induced and uninduced. (d) 17.5 hr after agitation for the blank and 18 hr after agitation for the induced and uninduced.

Figure 3. Microplate readings at 590nm for samples taken from the sedimentation assay using SoftMax Pro Software. Increase in absorbance at 150 min may have been due to the aggregation of the cells on the microplate prior to taking the measurement.

Conclusion

There is a significant difference between the aggregation of cells with the Antigen43 construct (BBa_K542009) and the cells without the construct after 18 hours. The pLacI promoter is known to be leaky. This was illustrated by the similarity between the uninduced and induced samples (Figures 2 and 3). These experiments support the conclusion that BBa_K542009 works as expected and the induced cells exhibit sedimentation.

• Aim – to verify Assembly Standard 10 compliance
• Method:

Escherichia coli bacterial culture containing plasmids BBa_K542009 (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_K542009 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_K542009

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.

• Conclusion:

The gel results for the pSB1C3- BBa_K542009 construct suggested that the sequence has a deletion of approximately 1.2 kb, when compared to the theoretical map. Additionally, this Biobrick contains an extra XbaI site. Thus we conclude that the BioBrick BBa_K542009 originally deposited with iGEM did not meet assembly standard 10.

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)

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