Team:TJU China/Notebook
Notebook
We determined the main project to be the new applications of the novel fluorescent protein smURFP. We came up with 3 directions one after another to rich our project. The first is to reform the protein structure. The second is to detect BV in bird’s blood. The third is to make intestinal bacteria tracking in vivo in real time come true. To achieve these goals, we thought deeply for best arrangement. For structure and BV detection, we need purified protein and BV. For intestinal tracking, we choose surface display and co-expression for different kinds of bacteria. For solving the problem of the membrane permeability of biliverdin, we selected the surface display the target protein with the biliverdin connected outside the bacteria in anaerobes or co-express the smURFP and the enzyme, HO-1, to produce the biliverdin inside the bacteria. We first chose 7 of the bacteria, which is of vital significant in human intestine, as our research subject.
1stweek
Detection BV and the Structure of smURFP
We read the paper of the targeted protein, smURFP, of Mr.Qian carefully, and exchanged the amino acid sequence for prokaryotic expression to get better effect of expression.
EHEC
We got the plasmids of PACYC184 and received the strain we need from YiGang Xu teacher
We designed the primers about surface display and sent to the company to synthesize
B. fragilis
We found a lot of paper and finally found a paper on the Cell magazine of a research team from Stanford University who did a similar work as we are going to do. Then our group had several deep discussion about that paper and decided to send an e-mail to Dr. Liz Stanely asking for the sharing of plasmid pWW3534, pWW3515, pWW3452, pWW3535, pWW3536 and the original pNBU2, also with some questions about the RBS they use in their designs. Besides, we studied the technique of molecular cloning in the lab.
L. Iactis
Enter the laboratory to learn the basic experimental skills of preparation for solid media and molecular cloning.
Design primers to align smURFP, RBS and HO-1 genes.
C. rodentium
We searched the reference to get proper plasmid. And then we found that we could use Pacyc184, the same with EHEC. In addition, we chose two promoter for alter. We also sent email for the bacterial strain but failed.
B. longum
We found a lot of material and paper, selected the anchoring sequences for bifidobacteria surface display, selected several promoters as backup, and developed a protocol for the construction of shuttle plasmids. Nevertheless we studied the technique of molecular cloning in the laboratory.
2ndweek
Detection BV and the Structure of smURFP
We got new complex gene of smURFP for prokaryotic expression, and of course, tried to pick up the gene to construct the plasmid with smURFP.
EHEC
We extract the plasmid from the glycerin bacterias of last year
B. fragilis
We got a reply from Dr. Liz Sranely of his promise to send us the plasmids. We designed primers for smURFP and HO-1, and also for the five anchoring sequences-INPN, INPNC, LPP-OMPA, Brka and AIDA. At the same time we continue to study in the lab and mainly the PCR.
L. Iactis
Obtain the expression vector pMG36e, L. Lactis strain NZ9000 and the culture method with the generous help of Nankai University
B. longum
We continued to study in the lab and actively promote our own research. We transformed the two plasmids from Jilin University into Escherichia coli. Three clones were picked and cultured respectively, and the plasmids were extracted successfully.
3rdweek
Detection BV and the Structure of smURFP
We successfully constructed the plasmid of PET-28b (+) with smURFP and transformed it to the bacteria for expression, E.coli BL21.
EHEC
We started the first round of PCR to get the target fragments of four kinds of surface display; and then continued the second round of PCR to connect two fragments. We succeeded in completing the connections of some fragments, but some failed. Now some surface display is undergoing the third round of PCR and the others are still continue the second round.
B. fragilis
We activated five types of E.coli with our anchoring sequences in their plasmids. Then the plasmids with anchoring sequences are extracted respectively and are ready for doing the first round of PCR experiment. Also, we add His tag in our primer and found proper restriction enzyme cutting sites according to the paper.
L. Iactis
2 rounds of Overlap PCR to obtain our target gene.
Activate E. coli containing pMG36e and extract the plasmid after amplification.
Transform shuttle plasmid pET28bs into DH5α, and extract the plasmid.
B. longum
Because we cannot obtain the synthesized anchoring sequence, we modified the entire protocol and designed the corresponding primers. At the same time we have selected the appropriate kit for extracting the genome of the strain.
4thweek
Detection BV and the Structure of smURFP
We tested the inducing conditions and the conditions of purification to get more yield of smURFP. And luckily, we got the purified smURFP.
EHEC
We started the first round of PCR to get the target fragments of four kinds of surface display; and then continued the second round of PCR to connect two fragments. We succeeded in completing the connections of some fragments, but some failed. Now some surface display is undergoing the third round of PCR and the others are still continue the second round.
B. fragilis
We activated five types of E.coli with our anchoring sequences in their plasmids. Then the plasmids with anchoring sequences are extracted respectively and are ready for doing the first round of PCR experiment. Also, we add His tag in our primer and found proper restriction enzyme cutting sites according to the paper.
L. Iactis
2 rounds of Overlap PCR to obtain our target gene.
Activate E. coli containing pMG36e and extract the plasmid after amplification.
Transform shuttle plasmid pET28bs into DH5α, and extract the plasmid.
B. longum
Because we cannot obtain the synthesized anchoring sequence, we modified the entire protocol and designed the corresponding primers. At the same time we have selected the appropriate kit for extracting the genome of the strain.
Preparing for the final examinations.
1stweek
A week off. A few members are preparing for some reagents.
2ndweek
Detection BV and the Structure of smURFP
We tested several conditions for smURFP to get the crystal of smURFP.
EHEC
Measuring biomass of EHEC completed with a growth curve.
Competent cells of DH5α prepared.
Extracting standard plasmids.
Failing of constructing standard modules of LPP, INPNC, INPN, AIDA.
B. fragilis
First round PCR for AIDA, INPN, INPNC and LPP.
C. rodentium
We finished overlap PCR of co-expression and INPNC. And we dealt with the freeze-dried powder of Citrobacter rodentium following the direction while getting some infectious microbe in the access of culturing.
B. longum
Try to overlap GLBP with HU promoter. But it is hard to do that, so we failed several times.hree clones were picked and cultured respectively, and the plasmids were extracted successfully.
3rdweek
Detection BV and the Structure of smURFP
We sent the crystal to Shanghai for the information of the diffraction points of it. And then, Chen Wu started the work of analyzing the structure of smURFP.
EHEC
Cultivation of bacteria stored in glycerol and failing to extract plasmid pACYC184.
B. fragilis
Second and third round PCR for AIDA, INPN, INPNC and LPP.
L. Iactis
Amplify the Smurfp、HO-1 and linker（the RBS sequence from the PMG36e and papers①）
Overlap PCR get the Smurfp-linker- HO-1
Activated Glycerol DH5α that contain PMG36e
Pick the monoclonal colony and purify plasmid, however, the result of the agarose gel electrophoresis is blank.
C. rodentium
We finished overlap PCR of Brka. And we fall in the DH5α conversion of coexpression-Pet28a plasmid and INPNC--Pet28a plasmid.
B. longum
We changed the strategy which separate GLBP into two sediments to make overlap more easier. However it was a pity that PFU protease which produced by our own lab cannot do PCR very well with the goal sequence is over 2000bp. So the property of the production was bad for further experiments.
4thweek
EHEC
Linkage of lpp, INPNC and INPN to vector pET28-a confirmed.
Making an appointment of usage of confocal.
B. fragilis
Enzyme dygestion and lignation of the surface display construction.
Make Competent cell of E.coli DH5α λ-pir for amplification of plasimd pWW3536.
L. Iactis
Get the new Glycerol DH5α that contain PMG36e and repeat the same experiment as last week
Digest the PMG36e and Smurfp-linker- HO-1 with Xba I and Hind III,ligate them together
Transform them into DH5α，but the result was not as we expected.
C. rodentium
We tried to Double Digest and convert of coexpression-Pet28a plasmid, INPNC--Pet28a plasmid and Brka-Pet28a plasmid and Brka-Pet28a plasmid was finished.
The Citrobacter rodentium was identified and isolated from the mixed strain using EMB culture medium and subcultured.
B. longum
This week we modified the previous plan. After failing for two times, we decided to try a new method called seamless cloning which main advantage is no need of digestive sites, thus it can definitely help us save a lot of time. We made two programs about constructing surface display simultaneously: one is to connect the HU-meaningless sequence-smURFP and link them into the pGH-PMB1 vector, after finishing this transformation, we finally insert the GLBP sequence into the constructed vector to replace the meaningless sequence. The other option is to connect the three segments of HU, GLBP, smURFP with overlapping regions and directly into the PGH-PMB1 vector.
In the end, we succeeded in connecting the HU-meaningless-smURFP to the PGH-PMB1. In addition, we successfully construct the co-expression vector.
1stweek
EHEC
Co-expression confirmed via confocal.
Extracting plasmid pacyc184.
Extracting plasmid pacyc184.
B. fragilis
Repeat doing Competent cells.
L. Iactis
Prepare the new Erythromycin solution（50mg/ml）、 LB solid medium that contain Erythromycin（200mg/ml、300mg/ml）
Repeat the same experiment as last week，there are some special monoclonal colony，but the purify plasmids are also blank by agarose gel electrophoresis.
C. rodentium
Co-expression and INPNC were reconnected and converted and INPNC-Pet28a plasmid was finished. We tried to convert the DNA fragments to the pACYC184 plasmid.
B. longum
We finished our experiments about the construction of the plasmids. The co-expression vector and the surface display vector which GLBP as the anchoring sequence has been sequenced and the results are positive.
2ndweek
EHEC
Ligation product of co-expression and INPN to plasmid pacyc184 available. Sequencing analysis is processing.
B. fragilis
Considering that we may have little possibility to do this bacterium well, we regretfully suspended the plan.
L. Iactis
Considering that we may have little possibility to do this bacterium well, we regretfully suspended the plan.
C. rodentium
We tried to make up the competence of Citrobacter rodentium and succeeded. And we finished the INPNC-Pacyc184 plasmid.
B. longum
We read some references and learned that a kind of anchoring sequence called ACMA could be widely used in the Gram positive bacteria, so we decided to have a try whether it could function or not. And we successfully constructed the plasmid for ACMA.
Then we try to do electro transformation for the first time. But we cannot sure whether it was succeeded or not since after we extract the plasmids and do gel electrophoresis, nothing was visible on the gel.
3rdweek
Detection BV and the Structure of smURFP
We tried to product the protein, smURFP, and purified it from E.coli BL21. The first time, we tested whether smURFP can combine with Biliverdin (BV) and emit infrared fluorescence.
B. fragilis
Try to do electro transformation and verification for the second time. But this time there were no monoclone on the plate.
4thweek
Detection BV and the Structure of smURFP
We setted a seires of ratio of smURFP and BV to test which condition is the best for emit infrared fluorescence.
EHEC
Preparing competent cells of EHEC.
Transforming vectors to EHEC failed.
Construction of standard modules.
C. rodentium
We failed in the coexpression-Pacyc184 plasmid and Brka-Pacyc184 plasmid.
We converted INPNC-Pacyc184 plasmid into the competence of Citrobacter rodentium, while see two kinds of bacterial colonies. So we used EMB culture medium to identified and isolated the Citrobacter rodentium.
B. longum
Try to do electro transformation and verification for the third time. This time we get the monoclone but we faced the problem we faced in the first time. We suspected whether the plasmid was into the bacteria or not. So we search the reference and modified the procedure.
1stweek
Detection BV and the Structure of smURFP
We collected the data of the test of the ratio of smURFP and BV, plot the relation schema of them and drew a conclusion.
EHEC
Regaining competent cells of EHEC.
Construction of standard modules.
C. rodentium
We finished the coexpression-Pacyc184 plasmid. And we converted INPNC-Pacyc184 plasmid into the competence of Citrobacter rodentium one more time and picked the monoclonal, made plate lithography, and extracted the plasmid to verify.
B. longum
Try to do electro transformation and verification for the fourth time. We realize that the ORI of B. longum had a low copy number, so it is not difficult to image why we always failed in extracting plasmid with 5 ml bacteria. Then we expand the culture quantity for more plasmid. But we still had difficulty in verification with enzyme digestion since the concentration were still not high enough.
2ndweek
EHEC
Testing fluorescence using Microplate Reader for E.coli BL21 of co-expression system.
Construction of standard modules.
C. rodentium
Changeing P2 on to the plasmid.
Construction of standard modules.
B. longum
Plasmid extraction for verification.
Construction of standard modules.
3rdweek
EHEC
Using confocal microscopy for testing fluorescence of E.coli BL21.
Construction of standard modules.
C. rodentium
Plasmid extraction for verification (with new promoter P2).
Construction of standard modules.
B. longum
Construction of standard modules.
4thweek
EHEC
Try to transform plasmid into new competent cells of ATCC35150.
Construction of standard modules.
C. rodentium
Plasmid extraction for verification (with new promoter P2).
Construction of standard modules.
B. longum
Construction of standard modules.
1stweek
The National Day！
2ndweek
EHEC
Regaining competent cells of EHEC.
Doing in vivo experiments on mice in Nankai University using engineered E.coli BL21.
Construction of standard modules.
C. rodentium
Try to do western blot. But nothing is on the gel.
Construction of standard modules.
B. longum
Construction of standard modules.
3rdweek
EHEC
Doing western blot of E.coli BL21 with plasmid carried INPNC suface display construction.
Construction of standard modules.
C. rodentium
Construction of standard modules.
B. longum
Construction of standard modules.
