Composite

Part:BBa_K3823014

Designed by: Yiying Zhang   Group: iGEM21_Tongji_China   (2021-10-02)


T7-SQR-J23110-SDO-AprBA-SAT

Tongji_China 2021 hope to solve the odor problem of waste food. According to the background we have researched, we found that hydrogen sulfide and ammonia are the two main odors that cause malodor in food waste, so we decided to convert these two malodor-emitting odors into non-toxic and odorless substances, so that we could solve the problem through engineered bacteria. After literature review and intense discussions, we decided to converse hydrogen sulfide into sulfate(figure 1)

Figure 1. pathway of sulfide oxidization

We choose SQR, SDO, AprBA, SAT to oxidize sulfide, using J23110 as the promoter. But the result is not satisfying, so we decided to use the stronger T7 promoter for SQR gene. Further optimization of promoter will be done in the future.

Construction

Figure 2.construct the plasmid with four genes
Figure 2.T7-SQR-J23110-SDO-AprBA-SAT

Characterization

1.The mRNA expression level of the target gene was detected by RT-qPCR
 We extracted RNA of our engineered bacteria and wild-type bacteria for RT qPCR experiment. The relative content of target mRNA in each group was calculated based on 16s gene of E.coli. The experimental results can prove that our engineering bacteria can transcribe the mRNA of the introduced gene normally. (There is no target gene in the wild type, the relative expression of engineering bacteria is very high and the difference is large)

Result of RT-qPCR for four genes

2.The expression level of the target protein was detected by SDS-PAGE
 We first tried to introduce plasmids linked with 1/2/3/4 target genes into E.coli DH5α, and detected their protein expression levels. However, due to the low expression amount and the limitation of Spectrophotometry, we did not find significant differences between the engineered strain and the wild type protein strips.

SDS-PAGE bands of four genes regulated by the promoter of J23110

Therefore, four target genes were added with efficient T7 promoter respectively and introduced into E.coli BL21(DE3).Under the condition of IPTG induction, all proteins of strain containing target genes and wild-type strain were extracted. SDS-PAGE experiment and Coomassie brilliant blue staining showed that the expression of each target protein could be realized in E.coli BL21(DE3).

SDS-PAGE bands of four genes regulated by T7 promoter

We had hoped to construct plasmids with T7 promoter for all four genes, and then test the protein expression, but the construction process was very difficult. Finally, due to the limited time, we connected the four genes, but only the SQR gene was regulated by the T7 promoter. In the staining results, only the SQR band was obvious, but it was speculated that the other proteins should be able to express normally.

SDS-PAGE bands of T7-SQR-J23110-SDO-APR-SAT

Protein function verification experiment

In view of the the limiting capacity of our laboratory to detect the intermediates in the sulfide oxidation pathway, we mainly verified the function of our engineered bacteria from the oxidation level of sulfide and the generation level of sulfate.
Characterization experiment of S2- oxidation amount
 We configured a series of sodium sulfide solutions with concentration gradient and tested them with detection reagents according to certain methods. The standard curve obtained is ideal. It can be considered that our detection method can accurately reflect the relative content of sulfide in the solution within this concentration range.

stander curve of S2-

We put the engineered bacteria and wild-type bacteria into a certain concentration of sodium sulfide solution, take out the bacterial solution every 30 min to detect the residual sulfide concentration. The results show that our engineered bacteria can oxidize sulfide better. (because the bacteria have a certain adsorption effect on sulfide, the initial sulfur ion concentration of the two groups of added bacterial solution is lower than blank)

concentration of S2- in liquid environment with different bacteria
SSAS: pET3a-T7-SQR-J23110-SDO-APR-SAT WT: wide type BK: blank

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 190
    Illegal XbaI site found at 47
    Illegal XbaI site found at 3581
    Illegal SpeI site found at 2054
    Illegal PstI site found at 1771
    Illegal PstI site found at 2983
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 190
    Illegal NheI site found at 151
    Illegal NheI site found at 1722
    Illegal NheI site found at 1745
    Illegal NheI site found at 2568
    Illegal NheI site found at 2591
    Illegal NheI site found at 3473
    Illegal NheI site found at 3496
    Illegal SpeI site found at 2054
    Illegal PstI site found at 1771
    Illegal PstI site found at 2983
    Illegal NotI site found at 1525
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 190
    Illegal BglII site found at 2254
    Illegal BamHI site found at 184
    Illegal BamHI site found at 3786
    Illegal BamHI site found at 5241
    Illegal XhoI site found at 1534
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 190
    Illegal XbaI site found at 47
    Illegal XbaI site found at 3581
    Illegal SpeI site found at 2054
    Illegal PstI site found at 1771
    Illegal PstI site found at 2983
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 190
    Illegal XbaI site found at 47
    Illegal XbaI site found at 3581
    Illegal SpeI site found at 2054
    Illegal PstI site found at 1771
    Illegal PstI site found at 2983
    Illegal NgoMIV site found at 255
    Illegal NgoMIV site found at 679
    Illegal NgoMIV site found at 1980
    Illegal NgoMIV site found at 4260
    Illegal NgoMIV site found at 4431
    Illegal NgoMIV site found at 5114
    Illegal AgeI site found at 1174
    Illegal AgeI site found at 2001
    Illegal AgeI site found at 2794
  • 1000
    COMPATIBLE WITH RFC[1000]


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