Team:IISc-Bangalore/Notebook
Notebook
The following page contains a weekly account of the team's activities in the lab.
Week 1
Basic buffers necessary for the summer session were prepared. Plates, tips and other equipment were autoclaved and kept ready, media prepared.
Week 2
DH5-α comp cells were prepared, and Biobricks BBa_K16530037, BBa_K731721, BBa_K525998, BBa_J18932, BBa_K1321337 were transformed into these. (transformation successful)
Week 3
Miniprep of transformed colonies, and digested plasmids were run on a gel. (positive bands)
Week 4
PCR products of 7 Biobricks with VF2/VR primers were run on a gel. (Bands of appropriate sizes were obtained)
Week 5
Assembly method 3 for imCherry was initiated- digest and extraction of T7 adapter backbone, ordered oligo, and BBa_J18932 with appropriate enzymes for three way ligation.
Week 6
Assembly continued- above fragments were ligated, transformed into DH5-α, transformants checked with colony PCR. Positive colonies were miniprepped, and transformed into E.coli BL21 (DE3).
Week 7
Colony PCR for BL21 (DE3) transformants performed. Protein extraction and purification, followed by SDS-PAGE, Ni-NTA column purification, and fluorescence characterisation for mCherry and imCherry were attempted- no protein obtained in case of imCherry.
Week 8
Phage resistant bacteria were identified for usage in the APES assembly and coevolution model. imCherry assembly was restarted. A part of interlab was done and reported. Phages were also isolated from culture and a new lysate was made, using which different methods of mutations were tried out for APES.
Week 9
Few more iterations of Interlab were carried out. imCherry assembly continued. Mutagenesis for APES and subsequent experiments were continued as well, despite unsuccessful attempts.
Week 10
Inter-lab work (CFU counting) was carried out. Extraction and amplification of the T7 adapter was done. imCherry assembly with T7 adapter was continued. Plaque assay performed, phage lysate obtained.
Week 11
Few more iterations of CFU counting carried out for inter-lab. Ordered DNA arrives. Mutation of phages with different mutagens for APES attempted.
Week 12
Ordered DNA was PCRed, digested, and ligated into digested linearised pSB1C3 backbone, and transformed into DH5-α . Transformants were tested with colony PCR, (negative results obtained) following which the above procedure was repeated. Interlab trials were repeated.
Week 13
Following negative results in second assembly trial, mcp-1 under T7 expression was reassembled. mCherry characterisation was initiated. Interlab trials continue.
Week 14
Mcp-1 under T7 expression, mcp-1+ HlyA and mcp-1+PelB assembly trials repeated. Colony PCR yielded positive results for mcp-1 under T7 colonies. Positive colonies were miniprepped, and digested, and bands of correct size were observed. mCherry was expressed, extracted, and SDS PAGE run. Ni-NTA column purification was done for mCherry, along with complete fluorescence analysis. Repeat of mcp-1+HlyA (under T4 recombination cassette), and mcp-1+PelB assembly.
Week 15
Transformation of the mcp-1 under T7 plasmid into BL21(DE3), and protein expression was done, followed by SDS-PAGE(unsatisfactory results were obtained). Repeat of imCherry assembly procedures initiated with the ordered fragment (Assembly method 1).
Week 16
Assembly procedures (verification of transformed plasmids with colony PCR, and EcoRI+PstI digest following miniprep) of imCherry continued, and positive results were obtained. Assembly of endolysin under constitutive promoter was begun, and positive results were observed after final E+P digest. mcp-1 under T7 expression was electroporated into BL21 (DE3), and following successful electroporation, protein expression was initiated.
Week 17
The process of assembling and preparing the various DNA fragments and making proper usable plasmids continued. (525998, 731721, MCP-1 + HlyA, MCP-1 + PelB). MCP-1 under T-7, whose plasmids were ready, was sent for sequencing. A successful sequencing was followed by attempts to extract the protein. Competent cells weren’t working, so new ones were made.
Week 18
The new competent cells appeared to work (at least DH5-α). The IDT supplied PelB + mcp-1 was tried upon for plasmid creation. The previous assemblies continued. Protein extraction for mcp-1 T7 was tried again and again with different conditions, but proper results not found. imCherry was ligated into an Ampicillin backbone.
Week 19
Previous assemblies continued. λ Red BioBrick assembly initiated. Tried optimisation techniques for MCP-1 T7. imCherry purification, and 6X-His purification initiated, and Endolysin protein expression work begun, following correct sequencing results. Plasmids (for gp37, gp38 and gp57) obtained from Dr Raiij.
Week 20
imCherry extraction and 6X-His purification continued. Previous assemblies continued. SDS-PAGE for mcp-1 along with endolysin run.
Week 21
Continued the previous assemblies. Continued working on gp57/gp38/gp37. mcp-1 extraction continued. Lytic activity characterization for Endolysin was initiated. Phage lysate extraction with BL21(DE3) and MG1655 were carried out.
Week 22
Previous assemblies were continued. Continued assembly of gp37 M-3 and M-4. Extraction of mcp-1 T7 continued. λ Red BioBrick assembly completed, and verified by a digest of the miniprepped plasmid.
Week 23
Work on mcp-1 T7 with Lambda red assembly continued. Meanwhile phage DNA extraction was also done. Electroporation for transformation of DH5α done. SDS PAGE yielded vague results, inclusion bodies suspected for this. Work on gp37 started, where two modifications M1 and M2 were looked into.
Week 24
Endolysin characterisation was done this week. Work on mcp-1 T7 was continued. Electroporation for imCherry was also done. A protocol for removal of inclusion bodies was also undertaken and gp37 work continued with modifications M1,M3 and M4.
Week 25
Dialysis of gp37 suspected to be in inclusion bodies and its treatment with urea was carried out. Folding assay(CD) was performed to verify proper folding. Triple transformations using the plasmids obtained from Dr Raiij was attempted. Assembly of mcp-1+HlyA/PelB under T7 initiated.
Week 26
Assembly of mcp-1+HlyA/PelB under T7 repeated on obtaining negative result. Triple transformation repeated. Assembly of gp37 M1,M3, M4 attempted.
Week 27
Assembly of gp37 M1, M3, M4 repeated. Triple transformation remains unsuccessful. Successful assembly of mcp-1 PelB under T7 expression system verified by E+P digest of miniprepped plasmid. Final mCherry-imCherry truncation profiles done.
Week 28
Successful assembly of mcp-1+HlyA under T7 expression system confirmed by correct sizes E+P digest of miniprepped DNA on gel.
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