Wet Lab work has not yet started. It will start in April.

Wet Lab work has not yet started. It will start in April.

Wet Lab work has not yet started. It will start in April.

Wet Lab work has not yet started. It will start in April.

Week 1: GH, TS, TK

• Antibiotic resistance:
  After having received our production strains S. blattae and R. planticola we could start lab work with testing them for natural antibiotic resistances. For this, they were grown in culture overnight and plated on LB-Agar plates supplemented with different antibiotics.

Week 2: TK, GH, LK, IF, MS, TS

• Antibiotic resistance:
  Due to inconsistent results in the week before, the test for antibiotic resistances had to be repeated. We additionally tested for resistance against chloramphenicol and used more different concentrations of the antibiotics and dilutions of the pre-cultures. The results showed, that all production strains have no antibiotic resistance for kanamycin. It can therefore be used as antibiotic resistance on our plasmids.
• Bacterial identification:
  To check the identification of our production strains, we performed a 16S rRNA PCR. All strains were correct, so glycerol stocks could be prepared.
• Competent cells:
  For the following experiments, we had to prepare electro competent cells of our productions strains and E. coli DH5α. This was done first for E. coli DH5α according to protocol.

Week 3: IF, NE, ME, MS, TK

• Competent cells:
  Electrocompetent cells for S. blattae and R. planticola were prepared.

 Week 4: IF, LK

• Test transformation:
  To check if our prepared electro competent cells were indeed electro competent, we transformed them with a simple vector containing a kanamycin resistance according to protocol and grew them on plates over night. Unfortunately, only E. coli DH5α showed resistance against kanamycin, suggesting that the transformation or the preparation of competent cells was not successful for the others.

Week 1:

• No wet lab work done.

Week 2: IF, LK, NE, TS, GH

• Test transformation:
  The previously prepared cells of our production strains were again tested for electro competence. The protocol was slightly changed using SOC medium instead of LB-medium and incubating the bacteria at their temperature optima (R. planticola: 30 °C, others: 37 °C). However, the cells again did not grow, showing that they were not electro competent.

Week 3: NE, TK, GH

• Preparation:
  For the following week, large amounts of media had to be prepared.

 Week 4: NE, GH, MS, AB

• Competent cells:
  The preparation of competent cells was repeated for S. blattae and R. planticola. The cells were again incubated at their respective temperature maxima.

Week 1: IF, LK, TK, GH, ME, AB

• Test transformation:
  

  The newly prepared electro competent cells were tested and compared with a test transformation of cells provided by our supervisor Genis. Only the S. blattae cells showed antibiotic resistance.

• MutB construct:
  After having received the synthesized constructs from IDT, we could start to prepare the constructs. MutB had to be ordered with a restriction site and synthesized in two fragments due to its length. Therefore, it had to be digested with HindIII, purified and ligated.

Week 2:

• No wet lab work done.

Week 3: AB, ME

• Competent cells:
  

  The preparation of electro competent cells was repeated for R. planticola and test transformations with a simple kanamycin vector were successful.

• MutB construct:
  The ligation of the two fragments was repeated. Additionally, the synthesized MutB1 and MutB2 fragments were amplified by PCR, followed by gel electrophoresis and gel extraction to prepare a stock.

Week 4: AB, ME

• MutB construct:
  The amplified MutB fragments were digested with HindIII, purified and ligated. However, gel electrophoresis showed that none of the ligations worked yielding products of wrong sizes.

Week 1: AB, ME

• MutB construct:
  

  Restriction, purification, ligation and gel electrophoresis had to be repeated, but the results were again negative.

• GlmS and BtuF constructs:
  The synthesized constructs were cloned into the pBAD202 D-TOPO vector.  In contrast to the suggested protocol, we used only half of the amount of the vector. The transformation was performed according to protocol into TOP10 E. coli. The resulting colonies were transferred into liquid culture and incubated over night. From the grown cultures, plates for storage were prepared and plasmid preparations were done.

Week 2: AB, ME

• MutB construct:
  

  Due to the difficulties of ligating the two fragments of MutB, we tried a different approach and prepared fragments with overhangs by PCR using newly ordered primers. After gel electrophoresis and purification, the fragments were directly ligated.

• BtuF construct:
  To check if BtuF was correctly inserted into pBAD202 it was digested with SmaI. The gel after electrophoresis did not show any bands.

Week 3: ME, TS, TK, MS

• MutB construct:
  

  To find out if the ligation was successful, a PCR to recover the fragments was done with the respective primers.

• GlmS construct:
  To check if GlmS was correctly inserted into pBAD202 it was digested with AccI. The gel after electrophoresis did not show any bands.

Week 4: TS, TK, MS, AB

• MutB construct:
  Due to problems with ligating the two MutB fragments with help of overhangs, we tried another approach. For this, the fragments were prepared with blunt ends via by PCR using respective primers. After gel electrophoresis and gel extraction the PCR products were then ligated via blunt-end ligation. However, the gel did again not show bands for products of the right size.

Week 5: TS, TK, MS, AB

• MutB construct:
  PCR, gel extraction and ligation of MutB blunt end fragments was repeated. To increase the amount of DNA, the product was amplified by PCR. Additionally, we tried to phosphorylate the MutB2 blunt end fragment before ligation.

Week 1: TK, AB, MS, GH, ME

• MutB construct:
  The ligation from last week was purified and analyzed by PCR and gel electrophoresis. However, we again did not get a product with the right size.
• GlmS and BtuF constructs:
  Cloning of the two constructs into the pBAD202 according to the D-TOPO protocol was repeated, followed by transformation of TOP10 cells. After growing the cells, plasmid preparations were performed.

Week 2: ME, MS, GH, AB, TK

• GlmS and BtuF constructs:
  The plasmids from last week were digested with respective restriction enzymes and new liquid cultures for both constructs were prepared. But none of the prepared plasmids showed the right restriction pattern after digestion.
• MutB construct:
  MutB1 blunt end was prepared by PCR and gel extraction and amplified by PCR. Additionally, MutB1 and MutB2 were again generated with overhangs by PCR. The PCR was not successful, so we tried another approach by following the protocol for overhang generation used by Harrison et al.

Week 3: AB, TK, GH, MS, TS, LK

• GlmS and BtuF constructs:
  Cloning of GlmS and BtuF into pBAD202 and transformation into TOP10 cells had to be repeated. The transformation was then plated on plates with minimal M9 medium, but no colonies grew.
• MutB construct:
  The generation of MutB1 and MutB2 with overhangs and MutB1 with blunt end was repeated. Only MutB1+blunt end could be produced. Additionally, a repetition of the PCR of MutB2 with the phosphorylated MutB2 was done, but MutB2 could not be amplified.

Week 4: GH, LK, ME, TK, TS, IF, MS, AB

• MutB constructs:
  The MutB+blunt end DNA was amplified by PCR, but the control gel was showed negative results.
• Preperation:
  Our LB-Kan and M9-Kan plates were tested with a transformation of PLP03 in DH5α and compared with plates provided by the lab. After negative results, our plates had to be discarded but plasmid preparation could be performed from clones of the control plates. We prepared new plates for us.
• GlmS and BtuF constructs:
  The constructs could be successfully amplified with the newly designed primers and GlmS and BtuF in pBAD202 were transformed into TOP10. The transformation for GlmS was successful and plasmid preparation could be performed.

Week 1: AB, ME, MS, GH, LK

• GlmS construct:
  A control of the GlmS plasmids was done by digestion and the constructs were additionally sent in for sequencing. All had the right sequence.
• BtuF construct:
  Liquid cultures of BtuF colonies in LB and RM medium were prepared and plasmid preparations were performed. The plasmid from the RM medium could be digested with SmaI, but the gel showed only an empty vector.
• MutB construct:
  The MutB fragments were amplified with the improved primers by PCR and the PCR products were purified and digested with BamHI. The ligation afterwards was successful and a gel extraction was performed.
• Competent cells:
  New electro competent cells for E. coli DH5α were prepared.

Week 2: LK, GH, AB, TK, ME

• MutB construct:
  We tried to amplify the ligated MutB, but the amplification did not work. Therefore, the ligation was repeated. PCR proved the successful ligation. MutB was then amplified by PCR and purified by gel extraction.
• GlmS construct:
  GlmS was transformed into TOP10 and E. coli LMG194 cells. The TOP10 cells could afterwards be grown in RM medium for the following expression study which was done according to the pBAD202 D-TOPO manual. Additionally, the GlmS construct was modified excluding the thioredoxin sequence (GlmS w/o Thio) and amplified by PCR.
• BtuF construct:
  The transformation of TOP10 with BtuF was unsuccessful and had to be repeated. After DNA preparation, the product was digested with SmaI.
• Test transformation:
  The newly prepared E. coli DH5α cells were checked for electro competence and the preparation of the kanamycin vector PLP03 worked.
• E. coli LMG194:
  Electro competent cells of E. coli LMG194 were prepared and tested by transformation with kanamycin vector.

Week 3: GH, LK, IF, MS

• MutB construct:
  MutB in pET101 was transformed into TOP10, the DNA prepared and sent for sequencing.
• GlmS construct:
  A Western Blot of the expression study was done. Additionally, a Colony PCR of the transformed cells from last week was performed to check that the construct is not empty and afterward glycerol stocks could be prepared. The rest of the colonies were grown in LB medium with kanamycin and Vitamin B₁₂.
• BtuF construct:
  BtuF in pET101 was transformed into TOP10 and the DNA prepared.
• BtuF and GlmS w/o Thio constructs:
  The constructs without thioredoxin were purified by gel extraction and transformed in pBAD202 into TOP10 and the DNA was prepared.
• MutB w/o Thio construct:
  The PCR for the preparation of MutB without thioredoxin was also repeated but again did not work.
• MutB, BtuF and GlmS constructs:
  To all three constructs a CACC(ATT) stop codon was added by PCR.

Week 4: GH, LK, IF, ME, TS, AB, MS

• MutB, BtuF and GlmS constructs:
  All constructs were amplified with new primers for the vector pBAD18. After gel extraction, double digestion and purification, GlmS and BtuF could be ligated in pBAD18. The constructs were then transformed into TOP10. For BtuF, no plasmids could be detected after plasmid preparation.
• MutB construct:
  pET101 with MutB was digested as a control and could be transformed into TOP10. After preparation, the DNA was sent in for sequencing.
• MutB constructs from the lab:
  Due to our difficulties with acquiring a correct MutB construct, we received old constructs from the lab. A plasmid preparation was performed for the samples PTM3A, PTM3B, PTML and PMB5.
• BtuF construct:
  pET101 with BtuF was digested as a control.
• GlmS construct:
  GlmS in pBAD202 was transformed into S. blattae and R. planticola. After plasmid preparation, only the plasmid from S. blattae could be digested and a glycerol stock prepared. For samples from R. planticola no plasmids could be detected.
• BtuF and GlmS w/o Thio constructs:
  pBAD202 with the respective constructs was digested as a control and three of the samples were analyzed through expression studies in E.coli TOP10. Expression of BtuF w/o Thio could be shown.
  

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  Western Blot that shows bands at the expected size of TorA-BtuF w/o Thio protein in E. coli Top10.

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  The samples were additionally sent in for sequencing.
  GlmS w/o Thio in pBAD202 was transformed into S. blattae and R. planticola.
  The constructs were also ligated into the pBAD18 vector, transformed into TOP10 and plated on LB plates with ampicillin and vitamin B₁₂.
• B₁₂ Assay:
  Guttman medium for the following B₁₂ Assays was prepared.

Week 5: GH, LK, IF, ME, TS, AB, MS

• Competent cells:
  New electrocompetent cells of R. planticola and S. blattae were prepared.
• BtuF and GlmS w/o Thio constructs:
  Sequences of constructs in R. planticola were all positive.
  BtuF in pBAD202 was transformed in R. planticola. The transformation was successful; colonies were picked the next day.
  Expression studies were performed for BtuF in S. blattae and GlmS in S. blattae and R. planticola.
• MutB constructs from the lab:
  The constructs PTM6, PTM3A and PTM3B were transformed in S. blattae and DH5&alpha, PMB5 was transformed in R. planticola and DH5&alpha and colonies were picked the next day. From the resulting liquid cultures, glycerol stocks were prepared and plasmid preparations were performed.
• MutB, GlmS and BtuF constructs:
  The constructs were amplified with new primers to add restriction sites for cloning in the iGEM vector pSB1C3.
• B₁₂-Assay::
  A test run of the assay was successfully performed using only defined concentrations of B₁₂.
• Successful expression of TorA-BtuF w/o Thio protein in E. coli Top10.

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  Western Blot that shows bands at the expected size of TorA-BtuF w/o Thio protein in E. coli Top10.

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Week 1: GH, LK, IF, ME, TS, AB, MS

• BtuF and GlmS w/o Thio constructs:
  The expression studies for BtuF in S. blattae and GlmS in S. blattae and R. planticola were analyzed with SDS page and western blot. Expression of BtuF in S. blattae could be shown (see figure 4). An expression study of BtuF in R. planticola was performed. Analysis with SDS page and western blot showed a successful expression (see figure 4).
  Plasmid preparations of the liquid cultures from the transformation of BtuF in R. planticola were performed.
  A control digestion of BtuF in R. planticola was performed and successful for all tested clones.
• Collaboration with Team TU Darmstadt:
  Transformations of S. blattae with MiniCol_active, MiniCol_mut and pSB1A3(empty vector) were performed. All transformations were successful, the colonies on each plate were counted.
• GlmS and BtuF constructs:
  GlmS and BtuF were cloned in pSB1C3 and transformed in E. coli TOP10. Plasmid preparations were performed and the plasmids were sequenced.
• MutB constructs from the lab:
  Expression studies were performed for PMB5 in R. planticola as well as for PTM3A, PTM3B and PTM6 in S. blattae. Analysis with SDS page and western blot showed expression of PMB5 in R. planticola.
• B₁₂-Assay:
  R. planticola with Glms w/o Thio as well as with BtuF w/o Thio were cultured and induced with arabinose. After harvesting, fractions of cytoplasm, spheroplasm and periplasm were separated. Samples of the fractions were submitted to the B12 plate assay.

Week 2: IF, ME, LK, GH, AB, MS, TK

• MutB constructs from the lab:
  PMB-Amp was transformed in S. blattae and PMB-Kan was transformed in R. planticola. All MutB constructs were checked for restriction sites that may interfere with the ones present in iGEM prefix and suffix. It was concluded that there were too many to continue the work with these constructs.
• B₁₂-Assay:
  The plates showed no growth of E. coliDSM4261 where the samples were added. Instead the area around the samples were lighter than the rest of the plates. This might be due to the presence of buffer P in the samples killing the E. coli cells. Therefore, in all following B₁₂-assays, a desalting of the samples was suggested.
  S. blattae with Glms w/o Thio as well as with BtuF w/o Thio were cultured anaerobically and induced with arabinose. Samples before and after induction were analyzed by western blot. Expression could be detected for BtuF and GlmS (see figure 4). After harvesting, fractions of cytoplasm, spheroplasm and periplasm were separated. Samples of the fractions were submitted to the B₁₂ plate assay as well as to SDS page and western blot. BtuF could be detected in the spheroplasm and the periplasm (REFERENCE to WB 14.10.). In the plate assay, no B₁₂ could be detected.
  The empty vector pBAD202D/lacZ was transformed in E. coli TOP10 to be used as a negative control. Colonies were picked and plasmid preparations of the resulting liquid cultures were performed. Then a transformation of pBAD202D/lacZ in R. planticola and S. blattae was performed.
• MutB construct:
  A PCR to amplify the ligated MutB was performed, but no bands were visible on the analyzing agarose gel.
• TorA-RFP construct:
  Biobrick BBa_E1010 from the distribution kit (**highly** engineered mutant of red fluorescent protein from Discosoma striata) was transformed in E. coli TOP10. Colonies were picked and a plasmid preparation of the resulting liquid cultures was performed. TorA was amplified via PCR and restriction sites for cloning into pSB1C3 were added. The product was purified by agarose gel extraction. TorA and RFP in pSB1C3 were ligated into pSB1A3 according to the “3A Assembly” protocol from iGEM. The assembly product was transformed into E. coli TOP10.
• GlmS and BtuF constructs:
  GlmS and BtuF in pSB1C3 were prepared for shipment.

Week 3: GH, LK, MS, TS, AB, ME, IF, TS

• TorA-RFP construct:
  Colonies from the TOP 10 transformation were picked and the resulting liquid cultures were analyzed with fluorescence microscopy. No expression of RFP could be detected.
• B₁₂-Assay and expression test:
  S. blattae and R. planticola with Glms w/o Thio as well as with BtuF w/o Thio were cultured anaerobically and induced with arabinose. Samples before and after induction were analyzed by western blot. After harvesting, fractions of cytoplasm, spheroplasm and periplasm were separated. Samples of the fractions were submitted to the B₁₂ plate assay and the photometric assay as well as to SDS page and western blot. The same was done for aerobic cultures of S. blattae and R. planticola with Glms w/o Thio as well as with BtuF w/o Thio. These cultures were grown with B₁₂ present in the medium.
  

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  Both Western Blots show the expression of BtuF and GlmS in R. planticola, and BtuF in S. blattae

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• The plate assay gave no evaluable results. In the photometric assay, B₁₂ could be detected in the cytoplasm and periplasm of R. planticola and S. blattae. Additionally, the western blots showed expression of BtuF in the periplasm of R. planticola and S. blattae.