Difference between revisions of "Part:BBa K4252001"

Revision as of 10:44, 11 October 2022

phosphate exportation

As a phosphorus export element.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 426
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1174

Introduction

So far, only one type of P exporting protein, YjbB, has been reported in E. coli.The mechanism of phosphorus export by yjbB is still unclear. But based on experimental phenomena we choose YjbB as the phosphorus output element in our project.

Engineering

DNA fragments containing yjbB was amplified from E. coli MG1655 genomic DNA using the primers yjbB-5’/yjbB-3.The PCR fragments were inserted into the Xbal/Xhol sites of the vector pET-22b(+). And the vector was transformed into BL21.

Design

Functional validation method The yjbB-BL21 strain was amplified to about O D600=0.6 in L B medium containing 1 / 1000 ampicyl resistance, induced protein expression with 0.1mM IPTG for 2 hours, washed twice with phosphorus-free MOPs medium, and continued induction with 0m M IPTG to measure the supernatant phosphorus concentration over time (1-7 hours) and OD value (0.1 per increase).

Result

Figure 1: When BL21-yjbB and wild-type BL21 grow in LB culture medium until OD600 is about 0.7, IPTG was added until the final concentration was 2mM and induced for 2 hours. The bacteria were resuspended in 50ml phosphate-free medium, and 2mM IPTG was added to continue induction. Then, measure the change of phosphate concentration in the culture medium with the increase of time. During the whole induction process, the turbidity of bacteria in the experimental group hardly changed.

As shown in the figure above, the concentration of wild type group phosphate is basically maintained at a stable value. From the fifth hour, the phosphate concentration in the culture of the experimental group was much higher than that of the wild type

group, and increased significantly with time. It is proved that the phosphorus export element is feasible.

@@ Line 30: / Line 30: @@
 ==Result==
 <br>
-[[File: 1.png|500px|thumb|center|Figure 1: When BL21-yjbB and wild-type BL21 grow in LB culture medium until OD600 is about 0.7, IPTG was added until the final concentration was 2mM and induced for 2 hours. The bacteria were resuspended in 50ml phosphate-free medium, and 2mM IPTG was added to continue induction. Then, measure the change of phosphate concentration in the culture medium with the increase of time. During the whole induction process, the turbidity of bacteria in the experimental group hardly changed.]]
+[[File: The phosphorus concentration in the supernatant changed with time.png|500px|thumb|center|Figure 1: When BL21-yjbB and wild-type BL21 grow in LB culture medium until OD600 is about 0.7, IPTG was added until the final concentration was 2mM and induced for 2 hours. The bacteria were resuspended in 50ml phosphate-free medium, and 2mM IPTG was added to continue induction. Then, measure the change of phosphate concentration in the culture medium with the increase of time. During the whole induction process, the turbidity of bacteria in the experimental group hardly changed.]]
 <br>
 As shown in the figure above, the concentration of wild type group phosphate is basically maintained at a stable value. From the fifth hour, the phosphate concentration in the culture of the experimental group was much higher than that of the wild type
   group, and increased significantly with time. It is proved that the phosphorus export element is feasible.