Fluorescence and Chromoproteins

By: José Manuel Sálvador Lopez

Here you can find José's work on the BifC complementation of the Cpx system. Curious about his results? Read more about it here.

May

Week 1 (8th of May - 14th of May)
Wrote the Thesis Proposal and decided on the proteins I would use.

Week 2 (15th of May - 21st of May)
More writing on Thesis Proposal, more deciding on the proteins I would use.

Week 3 (22nd of May - 28th of May)
Decided the split sites for the proteins that will be tested. The decision was based on structural information. Prepared electrocompetent E. coli.

June

Week 4(29th of May - 4th of June)
Started work in Snapgene. The primers for amplifying mRFP, mVenus and mCerulean as well as the backbone were designed and ordered.

Week 5 (5th of June - 11th of June)
Received primers. Resuspension of the stock and preparation of working solutions.

Week 6 (12th of June - 18th of June)
Transformation of plasmids from the Distribution Kit containing fluorescent proteins. Performed extraction and purification of said plasmids.

Week 7 (19th of June - 25th of June)
Amplification of inserts and backbones for ligation. Transformation of cells with the obtained constructs. Cells producing the full mRFP, mCerulean and mVenus are ready.

July

Week 8 (26th of June - 2nd of July)
Primers and synthetic DNA has arrived. Amplification of zipper domains, anm2CP and Dathail for ligation. More electrocompetent E. coli were prepared.

Week 9 (3rd of July - 9th of July)
Amplification of backbone and leucine zippers, as more samples are needed.

Week 10 (10th of July - 16th of July)
Assembly of plasmids containing chromoproteins and fragments of chromoproteins and fluorescent proteins. Transformed them into E. coli DH5α and extraction of plasmids for restriction analysis and sequencing.

Week 11 (17th of July - 23rd of July)
The restriction analysis and the sequencing showed contamination of mRFP in a number of samples. The following samples were successfully obtained: carboxy and amino fragments of mRFP, carboxy and amino fragments of mCerulean, amino fragment of mVenus and carboxy fragment of anm2CP.

Week 12 (24th of July - 30th of July)
The contaminated samples were transformed again. Only the carboxy fragment of Dathail was successfully cloned and sequenced. Amplification of Ultramarine and sfGFP sequences and fragments. Assembly into plasmids and transformation into E. coli DH5α.

August

Week 13 (31st of July - 6th of August)
Extraction of plasmids for restriction analysis and sequencing. All the proteins and fragments were successfully cloned, except for the amino fragment of Ultramarine. The assembly and transformation was repeated for this fragment, as well as for the entire sequence and the amino fragment of Dathail. The fluorescence of the fluorescence proteins as well as the reassembly was detected through a plate reader. The chromoproteins do not produce colored colonies.

Week 14 (7th of August - 13th of August)
Holiday - Out of office.

Week 15 (14th of August - 20th of August)
Partly holiday, looked mostly at sequencing results for the remaining days and planned ahead for next week.

Week 16 (21st of August - 27th of August)
An alternative to leucine zippers was selected: MreB and EF-Tu. The plasmids expressing mRFP, anm2CP and Ultramarine were transformed into BL21 to try to achieve higher expression.

September

Week 17 (28th of August - 3rd of September)
Plasmids containing both halves of sfGFP and mVenus fused to the respective leucine zippers under the control of the PBAD promoter were successfully obtained.

Week 18 (4th of September - 10th of September)
Constructions of a single plasmid containing the split Venus, with the amino-terminal half fused to EF-Tu and the carboxyl-terminal part fused to MreB. The reassembly in vivo of split proteins through leucine zipper interaction was successfully observed.

Week 19 (11th of September - 17th of September)
Plasmids containing mRFP, mCerulean, mVenus, sfGFP and Ultramarine under the control of the pBAD promoter were successfully obtained. Plasmids containing the split mRFP (split at 155), mCerulean (split at 155) and anm2CP (split at 168) fused to leucine zipper and under the control of arabinose, were successfully obtained.

Week 20 (18th of September - 24th of September)
Different lysis protocols were tried to make the reassembly in vitro of sfGFP and mVenus work. The attempt was unsuccessful. The fluorescence of the split proteins was tested. Only the split sfGFP and Venus showed signal. New primers were designed to split mRFP at the position 168 and mCerulean at the position 172. The full anm2CP showed color, whereas Ultramarine and the split anm2CP did not show any. New primer were designed to split the anm2CP at the positions 154 and at 210.

October

Week 21 (25th of September - 1st of October)
A plasmid containing the dathail chromoprotein under the pBAD promoter, a plasmid containing the amino-terminal half of the Venus fused to EF-Tu and a plasmid containing the carboxyl-terminal half of the Venus fused to MreB were successfully obtained. Absorbance spectra was obtained for the fluorescent proteins, the chromoproteins and the split Venus and sfGFP. Finally, a growth experiment was carried out, where the signal generation time among the different fluorescent proteins was compared. Venus was identified as the fastest protein generating a signal, whereas Cerulean was identified as the brightest one.

Week 22 (2nd of October - 8th of October)
Plasmids containing the split mRFP, mCerulean and anm2CP at the new positions were successfully obtained. An experiment testing the reassembly of venus mediated by EF-Tu and MreB, both in vivo and vitro, was carried out with negative results for both conditions.

Week 23 (9th of October - 15th of October)
Absorbance and fluorescence spectra were determined for the fluorescent proteins and their split versions. Quantum yields were calculated from the results. The temperature effect on the maturation and the stability of the fluorescent proteins and their split versions was tested.

Week 24 (16th of October - 22nd of October)
Absorbance spectra were determined for the chromoproteins and their split versions. A new protocol for detecting maturation of fluorescence while ignoring transcription and translation was tested. Final maturation rates were obtained from this experiment.

Team:Wageningen UR/Notebook/Journal/Fluorescence