Part:BBa_K339001

This part was currently in the Registry as Part:BBa_J03008 as part of the 2005 Cambridge project but was resubmitted due to the lack of a sequence.

Mutant Maltose Binding Protein (malE31)

This mutant form of maltose binding protein contains a two amino acid mutation at amino acid positions 33 and 34 causing the protein to have difficulty folding properly.

Usage and Biology

This part can be used to test and characterize various stress promoters.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 435
  Illegal BamHI site found at 171
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 133
  

Characterization of malE31 and malE and their ability to misfold in the periplasm of Escherichia Coli

Characterization of malE and malE31 and their ability to fold through testing with the cpxR promoter

Protocol:

Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pCpxR coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then sub-cultured in 20 ml broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose(percent). This was incubated in the shaker for 12-14 hours and RFP output was measured using 555 excitation and 632 nm emission frequency.

Results

"http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Unititled-7.png"

Figure 2: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies

"http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/lineofbestfitCpxR.png"

Figure 3: RFP output produced by the CpxR-I13507 system when co-transfected with I0500-B0034-MalE (red) and I0500-B0034-MalE31 (blue) at different arabinose concentrations. RFP levels were measured at 555 nm excitation and 632 nm emission frequencies.

Discussion of Results and Conclusion

Figure 2 and 3 indicate the RFP output normalized with growth ratio (OD) at different levels of arabinose. Figure 1 shows that CpxR-I13507 is activated at the highest level when MalE31, the periplasmic misfolder, is expressed. This occurs around 0.2% arabinose concentration. Similar trends are observed in the case of MalE which is a periplasmic folder. MalE and MalE31 activate the system at different levels. MalE31 has similar trends to MalE but has a higher level of RFP expression. These results prove that MalE and MalE31 can both activate the CpxR system however, MalE31, which misfolds, activates it more rapidly and at a lower level of arabinose concentration compared to MalE. If the line of best fit is studied, it is seen that MalE has very minimal level of Cpx activation. Whereas, malE31 has a linear regression which flattens out as the system reaches its upper threshold of detection. Biologically, this could mean that the MalE31 is activated at levels that saturate the cellular chaperones and cause the system to reach its threshold level of proteolytic and chaperone activities. Another interesting pattern observed is the fact that when MalE is constructed with CpxR-I13507 on the same plasmid (Green), the cell RFP output is much lower compared to cells co-transfected with CpxR-I13507 and I0500-B0034 –MalE. This indicates that insertion of high copy plasmid also induces stress in the periplasmic region of the cell consequently inducing the activation of CpxR system.

Testing of MalE and MalE31 folding capabilities with literature established reporter constructs

Protocol:

We obtained degP and cpxR reporter constructs from Dr. Tracy Raivio's lab. These constructs contain the promoters upstream of lacZ. They were characterized with NlpE, an outermembrane lipoprotein that activates the Cpx pathway. We made TOP10 E. Coli competent cells with plasmids of these constructs and then transformed in MalE and MalE31 circuits with arabinose inducible promoters. We also transformed in the NlpE expression construct which we received fro the Raivio lab as well. The purpose of this assay was to confirm that promoters coupled reporters could indeed be induced by misfolding protein and to show that the various maltose binding proteins that we received were functional. From plates, we made 5 mL LB cultures and induced with 1uL IPTG for cultures containing the NLPE constructs. 75uL of X-Gal was also added to each culture. The cultures were then grown up overnight in a 30°C shaking incubator and observed for color.

Results

"http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/tubes.png">

Figure 1: Image of overnight cultures. From left to right: NLPE in cells with the degP reporter construct, NLPE in cells with the cpxR reporter, malE31 in cells with the degP reporter, malE31 in cells with the cpxR reporter, malE in cells with the degP reporter contruct and malE in cells containing the cpxR reporter.

Discussion of Results and Conclusion

Figure 1 indicates that malE31 is able to activae the cpxR and and degP promoers while malE is not. This allowed us to conlucde that these parts are working as expected. Although these parts are both entered in the registry, the sequences are not complete, so we are submitting new versions of them, constructed ourselves. Once we knew that these parts were functional, we went o to characterize them with our reporter constructs.

Characterization of the malE and maE31 folding abilities through testing with the degP promoter

Protocol

Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pDegP coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then subcultured in 20 ml LB broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose. This was incubated in the shaker for 12-14 hours and RFP output was measured using 555/632 nm.

"http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/DegPinduction.png"

This figure demonstrates that the DegP promoter activated with 15 different concentrations of arabinose. This diagram shows that the DegP promoter (K239000) is not particularly sensitive to misfolding proteins.

Discussion and conclusions

The figure shows that MalE and MalE31 express the DegP promoter in a similar fashion. This is slightly contradictory compared to the literature. The literature claims that the DegP promoter is upregulated in the case of protein misfolding, graph shown . Since MalE and MalE31 have been tested using other experiments described in this page, it is reasonable to conclude that K230009 is not very responsive to protein folding stress, that is , the DNA might not be consistent.