Overview of our part libraries
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For our different experiments, we had to clone different plasmids. We used parts from the iGEM registry as well as parts that we newly designed. Check out our libraries below!
The Lambda RED Recombination Plasmid
To create our Lambda RED Recombination Plasmid (BBa_K3514004), we needed three cloning steps. First, we cloned the DNA sequences of Exo, Beta and Gam into three separate plasmid backbone via GoldenGate cloning. They were then cloned into a second plasmid backbone where they were combined with each a promoter and a terminator. In the third backbone, all three promoter-gene-terminator constructs were combined to one.
Disclaimer: The promoter we used to create the finished recombination plasmid in the registry is not the one we used for our experiments. The promoter we used is also a variant of the T7 Lac promoter that is very well characterized in our facilities. However, it contains sequences that are not compatible with the iGEM registry. As we did not want to mutate this promoter without being able to characterize its mutations, we decided to use the promoter BBa_K2406020 for our contribution to the iGEM registry. In its function and sequence it is very similar to the promoter we used, it is already characterized in the registry and compatible so other teams can use our plasmid.
| Name |
Nr: |
Function |
| Promotor T7 Lac |
BBa_K2406020 |
T7 promoter with Lac1 operon for bacteria species with constitutive Lac repressors (e.g. e.coli BL21 DE3) |
| Terminator |
BBa_B1001 |
Ends transcription |
| Exo |
BBa_K3514001 |
Part of the Lambda RED Recombination System |
| Beta |
BBa_K3514002 |
Part of the Lambda RED Recombination System |
| Gam |
BBa_K3514003 |
Part of the Lambda RED Recombination System |
| Spacer |
BBa_B0040 |
Keep space between genes constructs |
| Lambda RED Recombination Plasmid |
BBa_K3514004 |
Contains the Lambda RED Recombination system |
Phage Engineering
To engineer our T7 bacteriophage, we designed the Lambda RED Recombination system on a plasmid (BBa_K3514004, see above). The sequences we wanted to insert were modified to specifically replace the Major Capsid protein of the T7 phage. Special recombination sites were added to the gene constructs to achieve this. These sequences were not added to the registry as they are only relevant for our specific project. However, the sequences are linked below for anyone who is curious.
| Name |
Nr. |
Function |
| Lambda RED Recombination Plasmid |
BBa_K3514004 |
Contains the Lambda RED Recombination system |
| Human Plasma Gelsolin |
Sequence |
LPS binding Plasma Protein |
| GSN 160-169 |
Sequence |
LPS binding site of Gelsolin |
| Peptide 19-2.5 |
Sequence |
Antimicrobial Peptide |
Safety Mechanism
To create our Safety Mechanism on a plasmid (BBa_K3514007), we needed two cloning steps. First, we cloned the DNA sequence of the Major Capsid protein of the T7 phage into a plasmid backbone via GoldenGate cloning. It was then cloned into a second plasmid backbone where it was combined with a promoter and a terminator.
Disclaimer: The promoter we used to create the finished safety plasmid in the registry is not the one we used for our experiments. The promoter we used is also a variant of the T7 Lac promoter that is very well characterized in our facilities. However, it contains sequences that are not compatible with the iGEM registry. As we did not want to mutate this promoter without being able to characterize its mutations, we decided to use the promoter BBa_K2406020 for our contribution to the iGEM registry. In its function and sequence it is very similar to the promoter we used, it is already characterized in the registry and compatible so other teams can use our plasmid.
| Name |
Nr. |
Function |
| Capsid |
BBa_K3514006 |
Major Capsid Protein of T7 bacteriophage |
| Promotor T7 Lac |
BBa_K2406020 |
T7 promoter with Lac1 operon for bacteria species with constitutive Lac repressors (e.g. e.coli BL21 DE3) |
| Terminator |
BBa_B1001 |
Ends transcription |
| Safety Plasmid |
BBa_K3514007 |
Major Capsid Protein of T7 bacteriophage on a plasmid. Safety mechanism for PHANGEL. |
Characterization
In order to make a useful contribution for future iGEM teams we decided to add new information to existing parts in the registry. We picked three closely related constitutive promoters (BBa_J23101, BBa_J23105, BBa_J23109) with a well documented difference in expression levels from the registry in order to characterize their expression strength in a Cell Free System. In order to design the required plasmids we also chose one terminator from the registry (BBa_B1001). mCherry was utilized as the expressed protein. The complex of promotor, gene and terminator was assembled via Golden Gate cloning. Expression strengths were determined based on fluorescence measurements.
