Overview
XJU-China has added 21 new documents to the registration page with new information. They are genes (sufB, thrC, mnmA, argF, rps) from the strains we collected.
We obtained the wild strains with good ability to tolerate saline and base environment, they are Brevibacterium casei G20, Bacillus haynesii P19, Micrococcus luteus R17, Halomonas bluephagenesis TD01, Halomonas campaniensis LS21 and Enterobactercloacae RS3. We find their function genes for the tolerance. Then we constructed 21 recombinant plasmids. We have tested sufB, and the sufB gene is very effective in improving the E. coli’s ability of function of model cells (E.coli).
Parts
The most important part is surfB(BBa_K4835001) from Halomonas campaniensis LS21. It has the good ability to enhance E.coli’s tolerance of saline and base environment. Here is the detail of this part.
sufB
1.Role, mechanism of expression
sufB is an iron-sulfur cluster scaffolding complex subunit, and iron-sulfur clusters may play an important role in bacterial salinity adaptation.
sufB is an iron-sulfur cluster scaffolding complex subunit, and iron-sulfur clusters are widely found in various organisms and are involved in electron transfer, substrate binding and activation, gene expression and regulation, etc. This gene may be used to improve saline and alkali tolerance in a variety of microorganisms, especially in model organisms.
Reference genome: H. campaniensis LS21 (H. campaniensis LS21)
Codon optimization
In order to verify that the target gene functions in the plant, the plasmid was constructed.
The selected vector is the sequence of E. coli expression vector pHT01 plasmid, and the recombinant plasmid was constructed with 5'Noc I and 3'Hind III as the digestion site.
The reaction system was at 37°C, and the digest was processed for three hours, and then the digest results were detected by agarose gel electrophoresis (1% agarose gel (containing 0.5 μg/mL of nucleic acid dye), 90 V electrophoresis for 30 minutes).
Observing the results of enzyme digestion, it was found that the enzyme bands corresponded to the length of the target gene, which indicated that the target gene was successfully constructed into the expression vector pET28a, and could be transformed in the following steps.
Plasmid transformation: preparation of receptor cells, introduction of plasmids into the receptor cells, and introduction of plasmids into the receptor cells.
Figure 2 Lecture for high school
Shake flask test, inoculate the induced bacterial broth at 1% inoculum into a 250ml conical flask (sample volume 100ml)
Shake flask test of salt concentration of E coli.Dh5 α-pET-28a (+)-LS21sufB
Nacl:1:02mol/L
Shake flask test of E coli. Dh5 α-pET-28a (+)-LS21sufB base concentration
PH11
