We purchased the iron bacteria from the Chinese Academy of Sciences strain library.Although we tried to culture the iron bacteria, we failed.
We decided to screen the iron bacteria from the nature,so we put the iron into the lack and hope that rust can form in a short time.
That’s a good news.The iron bacteria were screened from rusty iron sample and cultured by Winogradsky culture medium.
We start to construct quorum sensing part of the gene circuit. The DNA fragment,AfeR-GFP was synthesized by Genscript, and GFP was chosen as our reporter gene.
We were still busy with the construction of the recombinant plasmid. Because the difficulty of the work that we digested DNA fragment by restriction endonuclease and linked them with T4 ligase/SolutionⅠwas really beyond our imagination.
This we tried to use Gibson assembly to overcome the last week failure.And we did it!We have to say Gibson assembly is a real convenient and efficient way.
We characterized our interested fragment by detecting the fluorescence value of GFP under the different concentration of AHL, a signal molecule.
Our gene loop consists of many parts, and this week we constructed the expression vector of Dispersin B, whose promoter is inducted by the complex AfeR-AHL.
As mentioned above, Gibson assembly is more easy than the traditional method in construction work.However,we can’t use Gibson assembly in this part.That’s because there is Dispersin B’s coding gene in the genome of E.coli.So, you will see we would do the same thing in the following weeks until we succeed
We picked out some positive clones.Adding IPTG, culture overnight at 25℃.Then we broke these bacterial and obtained supernatant to do antibiofilm experiments and activity measurement.Also, the construction of enterobactin, a kind fo siderophore,was going on.
It was the hardest part we have ever constructed.Because the cloned fragment was so long that we have to carry out several rounds of PCR.
Our work was not going anywhere, so we asked for help from our instructor. With some discussion and attempt, we find some ways to solve problems.
Continue the work.
This week, we succeeded in inserting a gene cluster of enterobactin into vector pET-28a(+). After that, we get this supernatant to measure its ability of removing ferric iron.
We borrowed Light-on plasmid and its host bacteria,3J1 from Yang’s laboratory, and we thank Prof Yang Yi from East China University of Science and Technology for giving us the plasmid. We cultured 3J1 and measured growth curve.
We constructed Light-on-mCherry, and measured fluorescence intensity under different conditions.(7.29-8.4)
Due to the long period of expression of mCherry and some invalid data, we spent this week on doing the same thing as last week.Apart from this, we built a model by these data
Base on the last two weeks’ work, we construct Light-on-Mnase.
We divided these bacteria into two groups.One is cultured in the dark. The other is under the light.We measured OD600 to determine cell viability.
Build ,model about light suicide
We designed and synthesized three kinds of promoters, fur1,fur2 and fur3 which were inserted fur box.It cost some time to get these samples.
We characterize these modified promoters by measuring the fluorescence intensity of mCherry.
Based on the last week, we find that fur2 has the lowest leakage expression of lacI under low Fe concentration and the highest expression under high Fe concentration.Therefore we chosen fur2 promoter to use in our system.Then we constructed fur2-AD to realise that E.coli can express ceropin AD under high iron ion concentration.
We observed and evaluated the expression product, when synthesized AD is used as a positive control.
We construct fur2-lysin to achieve that E.coli can express lysin under IPTG induction.
We finished the characterization of lysin by measuring the light absorption of 600nm.Meanwhile, we also use normal temperature scanning electron microscope to observe the effect of lysin. In addition to this,the Effects of killing iron bacteria with antibacterial peptide, DspB and siderophore were considered comprehensively.