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Parts
We have designed and utilized several new composite parts consisting of elements that are not registered, in this form, on the iGEM parts registry, yet. This is one of our contributions to future iGEM-Teams especially those, that may also work in the field of biofilm degradation or other related areas.
| ID | Name | Type | New Part | Function | Status |
|---|---|---|---|---|---|
| BBa_K4575013 | Expression cassette for Mutanase | Composite | Yes | Methanol inducible expression and secretion of Mutanase in Pichia patoris | Fragment with overhangs exists |
| BBa_K4575015 | Expression cassette for Dispersin B | Composite | Yes | Methanol inducible expression and secretion of Dispersin B in Pichia patoris | Fragment with overhangs exists |
| BBa_K4575014 | Expression cassette for Dextranase | Composite | Yes | Methanol inducible expression and secretion of Dextranase in Pichia patoris | The part was divided into two fragments that exists with overhangs |
| BBa_K4575050 | Expression cassette for AMP BB-41 | Composite | Yes | L-Arabinose inducible expression in E. coli | Successful cloned in pBAB18, transformation in BW25113 E. coli strain, but not yet any successful expression |
| BBa_K4575051 | Expression cassette for AMP α-5 with enterokinase and bromcyan cleavage site | Composite | Yes | L-Arabinose inducible expression in E. coli | Successful cloned in pBAB18, transformation in BW25113 E. coli strain, but not yet any successful expression |
| BBa_K4575052 | Expression cassette for AMP α-5 and GFP with enterokinase and bromcyan cleavage site | Composite | Yes | L-Arabinose inducible expression in E. coli | Successful cloned in pBAB18, transformation in BW25113 E. coli strain, but not yet any successful expression |
| BBa_K4575053 | Expression cassette for polycistronic AMP consctruct with ninefold α-5 | Composite | Yes | L-Arabinose inducible expression in E. coli | Problems with ordering and never arrived |
| BBa_K4575054 | Expression cassette for polycistronic AMP consctruct with nine different AMPs | Composite | Yes | L-Arabinose inducible expression in E. coli | Problems with ordering and never arrived |
| BBa_K4575005 | AOX1 promoter | Basic | Yes | Strong, methanol inducible promoter for Pichia patoris | Part of BBa_K4575013, BBa_K4575014 and BBa_K4575015 |
| BBa_K4575007 | Alpha factor secretion signal | Basic | Yes | Secretion signal for yeast cells | Part of BBa_K4575013, BBa_K4575014 and BBa_K4575015 |
| BBa_K4575008 | Mutanase | Basic | Yes | Hydrolysis of alpha 1,3 glycosidic bonds of glucan polymers | Part of BBa_K4575013 |
| BBa_K4575009 | Dispersin B | Basic | Yes | Hydrolysis of beta 1,6 glycosidic bonds of glucan polymers | Part of BBa_K4575015 |
| BBa_K4575010 | Dextranase | Basic | Yes | Hydrolysis of alpha 1,6 and alpha 1,3 glycosidic bonds of glucan polymers | Part of BBa_K4575014 |
| BBa_K4575012 | His-tag | Basic | Yes | Tag for protein purification via IMAC | Part of BBa_K4575013, BBa_K4575014 and BBa_K4575015 |
| BBa_K3257011 | Spacer | Basic | Yes | A spacer between RBS and start codon | Part of BBa_K4575050, BBa_K4575051, BBa_K4575052, BBa_K4575053 and BBa_K4575054 |
| BBa_K4575019 | Glycine rich linker | Basic | Yes | Efficient ribosome binding site from bacteriophage T7 gene 10 | Part of BBa_K4575050, BBa_K4575051, BBa_K4575052, BBa_K4575053 and BBa_K4575054 |
| BBa_K4575020 | Enterokinase cleavage site Variant 1 | Basic | Yes | Cleavage site for enterokinase | Part of BBa_K4575050, BBa_K4575051, BBa_K4575052, BBa_K4575053 and BBa_K4575054 |
| BBa_K4575022 | Enterokinase cleavage site Variant 2 | Basic | Yes | Cleavage site for enterokinase | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575023 | Enterokinase cleavage site Variant 3 | Basic | Yes | Cleavage site for enterokinase | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575024 | Enterokinase cleavage site Variant 4 | Basic | Yes | Cleavage site for enterokinase | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575025 | Enterokinase cleavage site Variant 5 | Basic | Yes | Cleavage site for enterokinase | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575026 | Enterokinase cleavage site Variant 6 | Basic | Yes | Cleavage site for enterokinase | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575027 | Enterokinase cleavage site Variant 7 | Basic | Yes | Cleavage site for enterokinase | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575028 | Enterokinase cleavage site Variant 8 | Basic | Yes | Cleavage site for enterokinase | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575029 | Enterokinase cleavage site Variant 9 | Basic | Yes | Cleavage site for enterokinase | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575030 | AMP BB-41 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575050 |
| BBa_K4575031 | AMP α-5 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575051, BBa_K4575052, BBa_K4575053 and BBa_K4575054 |
| BBa_K4575032 | AMP GH12 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575033 | AMP α-10 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575034 | AMP BB-33 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575035 | AMP BB-34 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575036 | AMP α-11 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575037 | AMP FBα-20 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575038 | AMP α-7 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575039 | AMP BB-54 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575040 | Cyanogen bromide cleavage site | Basic | Yes | AMP against S. mutans | Part of BBa_K4575050, BBa_K4575051, BBa_K4575052, BBa_K4575053 and BBa_K4575054 |
| BBa_K4575041 | Linker | Basic | Yes | Improvement of flexibilty and folding | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575042 | Linker | Basic | Yes | Improvement of flexibilty and folding | Part of BBa_K4575053 and BBa_K4575054 |
| BBa_K4575044 | GFP | Basic | Yes | Improvement of translation and protein folding | Part of BBa_K4575052 |
| BBa_K4575062 | AMP α-5 Variant 2 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 |
| BBa_K4575063 | AMP α-5 Variant 3 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 |
| BBa_K4575064 | AMP α-5 Variant 4 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 |
| BBa_K4575065 | AMP α-5 Variant 5 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 |
| BBa_K4575066 | AMP α-5 Variant 6 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 |
| BBa_K4575067 | AMP α-5 Variant 7 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 |
| BBa_K4575068 | AMP α-5 Variant 8 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 |
| BBa_K4575069 | AMP α-5 Variant 9 | Basic | Yes | AMP against S. mutans | Part of BBa_K4575053 |
| BBa_K431006 | His4 | Basic | No | Coding sequence for histidinol dehydrogenase which is important for biosynthesis of histidin, thus often used for selection of positive transformants of His4 deficient Pichia pastoris cells | Fragment with overhangs exists |
| BBa_K3196026 | AOX1 terminator | Basic | No | Terminator of transcription derived from Pichia pastoris | Part of BBa_K4575013, BBa_K4575014 and BBa_K4575015 |
| BBa_K1486003 | Glycine-rich linker | Basic | No | Improvement of folding and fuction of proteins with His-Tag | Part of BBa_K4575013, BBa_K4575014 and BBa_K4575015 |
| BBa_K4046050 | Kozak sequence | Basic | No | Facilitates the proper initiation of translation in eukaryotic mRNA transcripts | Part of BBa_K4575013, BBa_K4575014 and BBa_K4575015 |
| BBa_K2936012 | araBAD promoter | Basic | No | L-Arabinose inducible promoter for E. coli | Part of BBa_K4575050, BBa_K4575051, BBa_K4575052, BBa_K4575053 and BBa_K4575054 |
| BBa_K3257011 | T7 RBS | Basic | No | Efficient ribosome binding site from bacteriophage T7 gene 10 | Part of BBa_K4575050, BBa_K4575051, BBa_K4575052, BBa_K4575053 and BBa_K4575054 |
Troubleshooting
During the course of the project, numerous experiments encountered challenges, which we endeavored to rectify through extensive research and literature review and changes in the experimental set up. We encountered significant difficulties during Colony-PCR and attempted various approaches documented in the literature, yet we were unsuccessful.
During troubleshooting for the Colony-PCR, we suspected that our cells were not lysing, thus impeding the success of the Colony-PCR and hindering the visualization of the desired bands. In the restriction analysis, we were able to identify many cells harboring our desired cloned DNA construct. Sanger sequencing further confirmed the success of our cloning efforts. However, the Colony-PCR failed to reveal the bands, despite consistent positive control PCR outcomes. Towards the end of the Colony-PCR troubleshooting, we made the decision to heat the cells at 98°C for 45 minutes, deviating from the conventional 5-10 minutes at 98°C in water or 20mM NaOH. This modification allowed us to visualize the desired bands. Therefore, we want to leave this method to the future iGEM teams so that they use the method when their cells do not lyse, and the Colony-PCR failed to yield bands. The Colony-PCR with 45 minutes at 98°C in water or 20mM NaOH, was performed akin to a standard PCR, including an additional lysis step. For the PCR, 1-2 µl of the lysed products were utilized, dependent on the size of the cell colonies picked.
A new approach to peptide synthesis in recombinant organisms and subsequent purification
In our research project, we employed antimicrobial peptides (AMPs) with the objective of expressing them within our cellular system, purifying them through affinity chromatography, and utilizing them against S. mutans. The primary challenge we faced pertained to synthesizing and investigating peptides of 10-15 amino acid length within the cells. The initial hurdle encountered in the course of the project was that our peptides were too small to be translated through ribosomes. Furthermore, the attempting to append an additional sequence, can alter the length, structure, potential efficacy, and effectiveness of the AMP, yielding unexpected results. To circumvent these challenges, we conceived an intriguing idea that we believe holds promise for synthesizing and purifying very small proteins or peptides without introducing extraneous unwanted amino acids.
To address the translation impediment, we incorporated GFP or other AMPs into our composite parts, allowing them to be translated as a protein of approximately 300 amino acids in length. However, this approach introduced a secondary concern: how to eliminate the surplus amino acids. To tackle this, we proposed utilizing a protease to remove the excess amino acids. To achieve this, we designed an Enterokinase cleavage site before our AMP, enabling the removal of all amino acids preceding the AMP. To remove the remaining amino acids and tags from the C-terminus of the AMPs, we considered employing cyanogen bromide and carboxypeptidase A. Given the absence of suitable known protease cleavage sites that could remove all amino acids following the C-terminus of the AMPs, we resorted to cyanogen bromide, a chemical molecule used to cleave proteins after methionine. Therefore, we appended a methionine at the C-terminus of the AMPs, allowing for the removal of all amino acids following methionine. Subsequently, carboxypeptidase A, an exopeptidase catalyzing hydrolytic cleavage of peptide bonds from the C-terminal end, was employed to eliminate the methionine. This enabled the purification of our desired AMPs at the correct length.
Regrettably, the expression attempts were unsuccessful, precluding us from demonstrating whether this method is effective in producing peptides or proteins smaller than 50 amino acids.