Part:BBa_K2449026
cenA-hlyA and secretion system with hlyB and hlyD regulated by penI
Type 1 secretion system, secreting the to enzymes endoglucanase and exoglucanase.
IT WAS NOT POSSIBLE TO DETECT ANYTHING.
Congo Red agar plates
To assess our cellulose degradation strains of E. coli we performed a screening with Carboxymethyl cellulose (CMC) Congo red agar plates. Congo red binds to cellulose molecules, making i possible to visualise the breakdown of the Congo red bound cellulose. NO visible difference between the bacteria this brick and the controls, suggesting that the type 1 secretion system is not working as intended.
Figure 1E. coli cultures containing seven different BioBricks plated on a Congo red plate containing the water soluble cellulose derivative CMC. Cellobiose phosphorylase on pSB1C3 with LacI promoter
SDS-PAGE of secreation
Secretion of the cellulases, endoglucanase and exoglucanase, was examined on a SDS-PAGE, with samples taken from the media isolated from an overnight culture. From the SDS-PAGE at Figure Y3 it was not possible to identify any proteins at the expected mass of endoglucanase and exoglucanase (47 kDa and 51 kDA, respectively)
Figure 2. Coomassie stained SDS-PAGE wíth Media isolated from five E.coli strains,
Conclusion
This BioBrick does not work! It is not possible to se any expression of the proteins.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2327
Illegal NotI site found at 627 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2179
Illegal BamHI site found at 3389 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 260
Illegal NgoMIV site found at 633
Illegal NgoMIV site found at 1135
Illegal NgoMIV site found at 2219
Illegal AgeI site found at 2315 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 680
Illegal SapI site found at 2027
Illegal SapI.rc site found at 763
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