"If you're a bird, I'm a bird"
This week we made a mastermix for the replication of the backbone and our G-blocks. All the ordered G-blocks were resuspended. We performed a PCR to amplify the amounts of G-blocks that we had available. Buffers and stocks were made for the agarose gel to check whether the PCR protocol was successful. See the Experiments page for the protocols.
This week we performed PCR for the amplification of the G-blocks that didn’t work last week. For the successful PCR products we performed a clean-up and checked the yield using Nanodrop (see figure 1). After the clean-up we set up a Golden Gate reaction and made LB plates with ampicillin. Additionally we made competent E.coli DH5α cells and the stocks required for the protocol. Finally we transformed the competent cells with the successful Golden Gate constructs, the transformation we checked using a colony PCR. See the Experiments page for the protocols.
| Name | [DNA] | A260/A280 | A260/A230 |
|---|---|---|---|
| R1AB6 nanobody monomer | 40.6 | 1.769 | 2.849 |
| Golden gate AmpR | 6.8 | 1.447 | 1.495 |
| Golden gate anti-GFP nanobody | 43.35 | 1.713 | 3.259 |
| Gibson AmpR | - | - | - |
| Gibson anti-GFP nanobody | - | - | - |
| pYTK001 entry vector | 5.3 | 1.337 | - |
Figure 1. Nanodrop concentrations for Golden Gate reactions.
This week we reperformed last week's colony PCR with a new touchdown protocol (see experiments for protocol). We redid the transformations for the initial experimental anti-GFP & anti-influenza entry plasmids. Then we PCR’ed the GFP3 anti-GFP nanobody and pTRKH3 expression vector, subsequently we performed a clean up and executed a Golden Gate reaction. For the Golden Gate we tested a new protocol (see figure 2), eventually discontinued the protocol in the future. Additionally we also performed a Golden Gate with the anti-influenza nanobody + Ampicillin resistance in the entry vector. From the anti-Influenza ligation we performed a transformation and checked it with a colony PCR and subsequent agarose gel.
| Name | Concentration |
|---|---|
| 10x T4 ligase buffer | 1µl |
| T7 ligase | 0,5µl |
| BsAⅠ/BmbⅠ | 0,5µl |
| Backbone (vector) | 20ng |
| R1AB6 nanobody monome | 20ng |
| Golden gate AmpR | 20ng |
Figure 2. New Golden Gate reaction mixture.
This week we tested the purified PCR product from the vector pTRKH3 & R1A-B6-AmpR in pYTK001 (see figure 3) with Nanodrop. We performed a transformation with anti-GFP nanobody in the expression vector pTRKH3. Additionally we started making MRS media for the cultivation of L.reuteri.
| Name | [DNA] ng/µl | A260/A280 | A260/A230 | |
|---|---|---|---|---|
| Plasmid | R1A-B6 | 100,9 | 1,865 | 2,247 |
| PCR | R1A-B6 + AmpR | 65,450 | 1,887 | 2,677 |
Figure 3. Nanodrop concentrations for the anti-influenza nanobodies.
This week we tried the initial protein expression experiment with IPTG induction.
This week we tested the protein mixture of last week's induction by using an SDS-PAGE gel 18% (see Experiments). Also this week we started cultivating L.reuteri, made growth curves of L.reuteri, and prepared competent cells. We tried the first electroporation (transformation) experiments with L.reuteri. Additionally we redid some E.coli transformations but this time using the strain BL21.
This week we tested the transformations of BL21 by means of a colony PCR. We started using vector pUC18 as a control vector to check transformation efficiencies, since our competent cells started to become less competent. Checked competency of both E.coli strain (DH5α & BL21). Finally we redid transformation with both E.coli strains and L.reuteri.
This week we focussed on the InterLab study. We prepared all the stocks and successfully performed almost all of the transformations in E.coli DH5α.
This week we redid some of the transformations of the Interlab study. Additionally we performed the last plate reading experiments for the InterLab study. For our main project we received newly ordered G-blocks for the kill-switch, which we multiplied. We cleaned up the PCR products, ligated the G-blocks and performed transformations on E.coli DH5α & BL21. Started cloning 4 different iterations of the anti-influenza nanobody (2 mono- & 2 bivalent) into the entry and expression vectors. Performed a promoter exchange on the construct pTRKH3 with anti-GFP. Finally we performed a colony PCR on transformed L.reuteri strains, from previous weeks, due to slow growth.
This week we redid L.reuteri transformations and checked the kill-switch constructs by means of a colony PCR. We send kill-switch products for sequencing.
This week we finished the promoter exchange and started with protein expression and purification. We did a transformation with the new promoter exchanged products and send kill-switch parts for sequencing after performing Nanodrop.
This week we transformed remaining kill-switch components into E.coli DH5α & BL21. We made a M9 medium for the kill-switch parts test experiments. Additionally we tested the kill-switch transformation with colony PCR and agarose gel, and sent constructs for sequencing. We performed the initial testing of the kill-switch fluorescence protocol. Finally we started rigorously testing the protein purification protocol.
This week we performed some of the kill-switch fluorescence experiments, and tested the constructs again using colony PCR. Redid transformations for some of the missing parts of the kill-switch constructs or if they were only available in either E.coli DH5α or BL21. Tried to optimize the L.reuteri transformation. Tried to optimize the protein expression and purification, performed Western Blot to check if protein was present.
This week we performed the last plate reader (fluorescence) experiments for the kill-switch. Additionally we performed large scale protein expression and purification for an ELISA experiment.
This week we colony PCR tested the last L.reuteri transformation MRS plates since some growth was detected, samples were put on gel and imaged.