Team:YAU-China/Parts
New parts
| Name | Type | Description | Length（bp） |
| BBa_K2999000 | Translational_Unit | RBS+PelA | 2947 |
| BBa_K2999001 | Translational_Unit | RBS+PslG | 1379 |
| BBa_K2999002 | Promoter | tac Protomer | 495 |
| BBa_K2999004 | Promoter | PA2069 Promoter | 1078 |
| BBa_K2999005 | Translational_Unit | RBS+PrtN | 345 |
| BBa_K2999007 | Composite | T7+malS | 2173 |
| BBa_K2999008 | Promoter | T7 promoter | 106 |
| BBa_K2999009 | Composite | Ptac+RBS+PelA+RBS+PslG | 4821 |
| BBa_K2999010 | Composite | PPA2069+RBS+PrtN | 1423 |
Data measurement
This part BBa_K2255000 is an enoyl-CoA hydratase, whose main function is to participate in the synthesis of cis-2-decenoic acid which can disperse the biofilm. Therefore, our team wants to verify the damage effect of the crude cis-2-decenoic acid solution after overexpression of the enoyl-CoA hydratase gene in Pseudomonas aeruginosa on the biofilm.
We cloned the enoyl-CoA hydratase and abundantly expressed in Pseudomonas aeruginosa lacking the DspI gene, using an empty plasmid as a control, and then extracting the crude extract of cis-2-decenoic acid, added different volumes of crude extract to culture biofilm, and observed the damage degree of biofilm.
Legend: Black is 40 μl of empty plasmid extract, gray is added with different volumes of cis-2-decenoic acid crude extract
Results: Cis-2-decenoic acid can destroy the biofilm effectively. With the increase of the volume of the extract, the destruction degree of the biofilm is greater. At 30-40 μL, the damage degree reaches the maximum value.
YAU-China team verified the function of this part in E.coli BBa_K523000 and got the following results:
Figure 1. The transformation results of laboratory sequence PA4110 related to this part
Figure 2. PCR agarose gel electrophoresis: 1, 2, 3 were white colonies, 4, 5 and 6 were blue colonies.
Then we tested the gene connection efficiency of the plasmid and the proportion of its positive colonies.
This is our transformation diagram of four groups of plasmids.
Figure 3.
Figure 4. Gene linkage data of BBa_K523000.
Figure 5. Statistical chart of correct number of positive clones.
Improve Parts
This year, we improved Plac + lacZ + malS (http://parts.igem.org/Part:BBa_K523006), which can produce starch hydrolase that can degrade starch. We used T7 promoter to replace lac promoter sequence on the protoplast, hoping to enhance the expression of starch hydrolase.
We designed the following experiments: The first line and the second line: Replacing malS gene with non malS gene PA1908 (Plac + lacZ + PA1908)；The third line and the fourth line: Transform with the original plasmid and test its function (Plac + lacZ + malS)；The fifth line: replace lac promoter with T7 promoter (T7 + malS).
We (YAU-China) monitored these three experiments in the same starch medium and cultured the cells for two days.
Figure. 1 shows the growth of cells in starch medium after two days；
Figure. 2 is the result of immediating the cells with iodine solution, which turns black immediately in case of starch；
Figure. 3 shows the result of iodine volatilization for 60 min.
We come to the following conclusion：
1. When the culture medium was immersed in iodine solution, the color of the first and second stripes was lighter than that of the third, fourth and fifth stripes (They have malS gene);
2. As time goes on, the medium becomes transparent after iodine volatilization;
3. After 60 minutes, halo appeared around 3, 4 and 5 stripes, and the halo size of 5 stripes was larger than that of 3 and 4 stripes.
References:
[1] Yuyama K T, Abraham W R. Cis-2-alkenoic acids as promising drugs for the control of biofilm infections[J]. Medicinal Chemistry, 2017, 12(999): 1-17.
[2] Amari D T, Marques C N H, Davies D G. The putative enoyl-coenzyme a hydratase DspI is required for production of the Pseudomonas aeruginosa biofilm dispersion autoinducer cis-2-decenoic acid[J]. Journal of Bacteriology, 2013, 195(20): 4600-4610.
[3] Davies D G, Marques C N H. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms[J]. Journal of Bacteriology, 2009, 191(5): 1393-1403.