Whats up in the lab!
Things are moving forward. Finally!
We are advancing a lot at the moment. We finally were able to complete our work with GFP-TAG-RFP, a biobrick BBa_K567018, which was a real pain to work with. However, the work really paid off and we were able to get good results from that line.
Our GFP-TAG-RFP controls. A fusion protein of GFP and RFP and a linker with TAG-stop codon inserted between. If the cells are capable of amber suppression, RFP will be expressed. E.coli cell strain 321.deltaA.exp together with pEVOL-pAzF in the presence of p-azido phenylalanine will read through the amber codon and insert the non-canonical amino acid into a genetically defined site at linker. Fluorescence measurements at 611 nm show more than 1000-fold signal of RFP in 321.A.exp compared to XL-1 Blue. The plate format was used to test effect of different concentrations of IPTG, arabinose and p-azido phenylalanine to expression levels.
Our cloning work with digoxigenin binder is done and the sequences have been verified by sequencing and test productions. This weekend we have optimized the production parameters to achieve the highest possible yields and we are now scaling up the production. We are aiming to get yields of about 10 mg/L. We will see how close we'll get.
If everything goes well with digoxigenin binder production and purification, we can start our assays! Our click chemistry reagents from Jena Bioscience have arrived and we can really start to concentrate on generating great results!
We will receive dengue binder sequences from IDT this week and we will continue working with them with the cloning team. The work should go much smoother now that we have all the equipment ready and we know what pitfalls to avoid.
-Pyry and the rest of Lab Team