Part:BBa_K5291029
pBBRMCS2-bcsA-bcsB
BcsA encodes a catalytic subunit of cellulose synthase, which is critical for initiating cellulose chain synthesis by adding glucose units from UDP-glucose. Its complex structure, featuring transmembrane segments and cytoplasmic domains, is a key consideration for ensuring efficient catalysis and interaction with other subunits. BcsB is a periplasmic protein that collaborates with BcsA to form a functional channel, facilitating the translocation of the growing cellulose chain across the membrane.
Usage and Biology
We constructed the plasmid pBBR1MCS2-bcsA-bcsB for cellulose production with the goal of carbon dioxide fixation.
Fig.1 The schema of pBBR1MCS-bcsA-bcsB.
Due to the challenges associated with primer placement at the extremities of the target sequence, we designed primers for colony PCR to amplify a 3,226 base pair (bp) fragment. The resulting agarose gel electrophoresis pattern confirmed the accuracy of the colony PCR, indicating successful amplification of the intended DNA sequence.
Fig.2 The agarose gel electrophoresis analysis of pBBR1MCS2-bcsA-bcsB(3226bp).
SDS-PAGE analysis of the induced cells after the construction of the expression vectors showed that the bcsA,bcsB proteins had the expected molecular weights.
Fig.3 SDS-PAGE analysis of the expression of bcsA (83 kd) and bcsB (95 kd) in E. coli BL21.
Then we also conducted a series of experiments to verify the effect ofbcsA-bcsB system.
1. Cellulose FT-IR Analysis
We used engineered and non-engineered bacteria as experimental and control groups for a three-day cellulose production culture. After three days, we filtered, separated and dried the cellulose contained in 30 ml of the bacterial solution for FT-IR analysis. The characteristic peaks of the infrared Fourier spectrum (FTIR) of cellulose are mainly distributed in the range of 3300-3500 cm-1 (-OH stretching vibration), 2900 cm-¹ (methyl or methylene stretching vibration), 1640-1650 cm-1 (water molecule bending vibration), 1500-1600 cm-1 (C-O stretching vibration), 1375-1425 cm-1 (methylene bending vibration) 1500-1600 cm-1 (C-O stretching vibration), 1375-1425 cm-1 (methylene bending vibration), and the regions of 1160-1200 cm-1 and 1100-1030 cm-1 (C-O-C skeleton vibrations) and other regions. The characteristic peaks were evident in the experimental group, whereas in the control group, due to the problem of very small yields, the characteristic peaks were measured to be unclear.
Fig.4 Comparison of cellulose FTIR analysis between experimental and control groups.
2. Congo red staining
When cellulose is present, Congo red forms a red complex with it, causing the medium to appear red. Since we have not been able to resuscitate Rhodopseudomonas palustris very well, we have temporarily tested it in E. coli BL21.
Fig.5 BL21/pBBR1MCS2-bcsA-bcsB,BL21,BL21/pBBR1MCS2.
The engineered bacteria BL21/pBBR1MCS2-bcsA-bcsB, as well as the individual E. coli BL21 and E. coli BL21 transformed with the empty vector pBBR1MCS2, were subjected to Congo red staining. The results showed that the engineering bacteria produced obvious red areas after staining, while E. coli BL21 alone did not show red, and there was a non-obvious red halo around the outer ring of E. coli transferred to the empty vector, thereby confirming the cellulose production capability of the engineered bacteria.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2282
Illegal PstI site found at 3431
Illegal PstI site found at 3464 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2282
Illegal PstI site found at 3431
Illegal PstI site found at 3464 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2282
Illegal BglII site found at 1527
Illegal BamHI site found at 1204 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2282
Illegal PstI site found at 3431
Illegal PstI site found at 3464 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2282
Illegal PstI site found at 3431
Illegal PstI site found at 3464
Illegal NgoMIV site found at 265
Illegal NgoMIV site found at 802
Illegal NgoMIV site found at 814
Illegal NgoMIV site found at 1040
Illegal NgoMIV site found at 3355
Illegal NgoMIV site found at 3985
Illegal AgeI site found at 1994
Illegal AgeI site found at 4065 - 1000COMPATIBLE WITH RFC[1000]
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