Team:TU Darmstadt/Project/Lab Journal

Lab Journal

Welcome to our Lab Journal!

After 26 weeks in the lab we are happy to show you how our project came to be.
We started in late April, week 17 of 2019, with basic laboratory-practices and then developed our skill set to include exciting methods like transmission electron microscopy (TEM)and FRET assays. To read more about the methods or parts we used, simply follow the links or have a look at our methods notebook and parts page.

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Week 17

We started our journey by preparing competent cells, both TOP10-cells and BL21-cells.

Week 18

We did a protein purification of Sortase A7M and of mCherry-LPETGG. Start of cloning mCherry-LPETGG into a pET24-backbone via restriction and ligation. Transformation of GGGG-mCherry into BL21 in preparation for the following week.

Week 19

Protein purification of GGGG-mCherry. Start of cloning sfGFP-Strep-tag II-SP and scaffold protein (SP) into pET24 via ligase&nbsp cycling reaction (LCR).

Week 20

Assessing functionality of Sortase A7M using mCherry-LPETGG and 3-Azido-1-propamamine, however our SDS-PAGE didn’t show the expected bands.

Week 21

Assessing the functionality of Sortase A7M using mCherry-LPETGG and 3-Azido-1-propamamine once more, but it still didn’t seem to be working out, so we tried assessing the Sortase A7M functionality in a Copper-Click-Reaction using 3-Azido-1-propamamine and 5-Fluorescein-Alkyne (5-FAM). Sadly, this didn’t work either.

Week 22

This week we tried linking mCherry-LPETGG to GGGG-mCherry using the Sortase A7M. We couldn’t really make sense of it since our SDS-PAGE failed.
We changed the cloning method for sfGFP-Strep-tag II-SP from LCR to restriction and ligation since we kept lacking colonies on our selection plates and also started cloning the CP-LPETGG via LCR.

Week 23

Linking mCherry-LPETGG with GGGG-mCherry using Sortase A7M but it seemed like the protein concentrations used were too low to show any bands.
This week was the start of our cloning of Sortase A5M using restriction and ligation.
We also changed the cloning method of sfGFP-Strep-tag II-SP from restriction and ligation back to LCR.

Week 24

Laying low in the lab since big parts of our team went to meet-ups with other iGEM teams in The Hague.

Week 25

Almost the same as the week before, but we went on a team-building trip to the Alps.

Week 26

Protein purification of GGGG-mCherry and Sortase A7M.
Our cloning of CP-LPETGG and sfGFP-Strep-tag II-SP switched to using Gibson Assembly instead of LCR as the method of choice.

Week 27

Assessing the stability and integrity of functionality of the Sortase A7M while stored at 4 ˚C.
Assessment of different induction parameters for the expression of CP-LPETGG followed by a protein purification.

Week 28

Finally, a positive sequencing for the Sortase A5M, followed by the establishment of a glycerol stock. Sortase A7M assay of CP-LPETGG with GGGG-mCherry and mCherry-LPETGG.
Linking both mCherrys finally worked AND showed the expected bands in our SDS-PAGE. We couldn’t really come to any conclusions regarding the linking of GGGG-mCherry to CP-LPETGG.
And our Copper-Click-Sortase A7M and mCherry-assay failed once more, due to using the wrong concentrators.

Week 29

This weeks Sortase A7M assay of mCherry-LPETGG with GGGG-mCherry didn’t work out once again, leaving us with loads of questions. We purified the Sortase A5M and more GGGG-mCherry, since we almost ran out of the latter one.
We also started several new clonings this week.
The cloning of mCherry with a TVMV-protease-cleavage-site via LCR, mCherry and Sortase A7M into pDESTara2 via Gibson Assembly, the cloning of the dual expression-vector with sfGFP-Strep-tag II-SP and CP-LPETGG into pTeT7con2 in two cloning steps and the cloning of Strep-tag-SP into pACYCT2 via LCR.

Week 30

We purified Sortase A7M again, as the assays were using up so much. The purification provided us with an unusually high yield and enough enzyme for the rest of the project. The assay of , GGGG-mCherry using Sortase A5M also left us with no clue as to what was going wrong.
Start of cloning mCherry without Tag into pTeTW3con2 via restriction and ligation, and CP without tag into pACYCT2 via Gibson Assembly.
We also started the in vitro assembly of our VLPs. We then ultracentrifugated the reaction products and prepared our samples for transmission electron microscopy (TEM).

Week 31

Protein purification of sfGFP-Strep-tag II-SP. Our first in vitro assembly of the VLPs. We tried a comparative assay of the Sortase A5M and Sortase A7M using CP-LPETGG and GGGG-mCherry, as well as finding the optimal calcium concentration for the Sortase A5M . Both SDS-PAGEs only showed negative results. Ugh.
We started the production of our VLPs in vivo in E. coli. The following ultracentrifugation did not give us usable results.

Week 32

Comparative assay of Sortase A5M and Sortase A7M at 10 mM Ca²⁺, it was negative. Protein purification of mCherry-LPETGG.
Finished the cloning of mCherry without tag in pTeTW3con2.
We attempted the in vivo production of our VLPs again, this time following the ultracentrifugation with size exclusion chromatography (SEC). We then inspected both these VLPs, and in vitro produced and heat treated VLPs via TEM.

Week 33

Started cloning Sortase A from iGEM Stockholm 2016 via Gibson Assembly.
We changed the cloning method of Strep-tag II-SP in pACYCT2 from LCR to restriction and ligation.
Protein purification of CP without tag.
We assembled and purified VLPs in vitro for gel chromatography, however the gel did not show any bands.

Week 34

Assay for the optimal enzyme to substrate ratio for both Sortase A5M and Sortase A7M. IT WORKED‼‼!

Week 35

We repeated the enzyme-substrate ratio test for Sortase A5M and Sortase A7M, this time it did not work. Switching from Gibson Assembly to restriction and ligation for the cloning of Sortase A from iGEM Stockholm 2016.
Protein purification of sfGFP-Strep-tag II-SP.
Start of cloning sfGFP and mCherry into the pTeTW3con2-backbone.
Start of cloning FLIPR-TEV-LPETGG into a pACYCT2-backbone via Gibson Assembly.
We conducted an experimental production of VLPs in E. coli with the dual expression vector pTeTW3con2. Unfortunately the purification failed due to insufficient sonification.

Week 36

Assay of Sortase A5M and Sortase A7M with varying calcium concentrations, the SDS-PAGE could not be assessed.
We also did another assay of CP-LPETGG and bovine serum albumin (BSA) to double check whether CPs already assemble in high concentration of other protein in their surrounding.
Another attempt at cloning Sortase A from iGEM Stockholm 2016 via Gibson Assembly. Positive sequencing and creating of a glycerol stock of mCherry with a TVMV-protease cleavage site.

Week 37

We tried to make a Western-Blot of all previously used proteins. Unfortunately, it smeared all over the place and we couldn’t really make good use of it.
We also double checked, whether the sortase is responsible for linking VLP-components into multimeres, which are not able to assemble into proper VLPs. It showed that sortase does build multimers.
We changed the cloning of mCherry and Sortase A7M in pDEStara2 to insert sfGFP and Sortase A7M into pDESTara2.
Start of cloning TEV-site-polyG-sfGFP-SP. into pET24 and expression of TEV-site-polyG-sfGFP-SP.
For our collaboration with the Paul-Ehrlich-Institute (PEI) in Langen we assembled and purified VLPs both in vivo and in vitro.

Week 38

Repeating last weeks Western-Blot since it didn’t work
Finished the cloning of sfGFP and mCherry into the pTeTW3con2-backbone.
Protein purification of TEV-site-polyG-sfGFP-SP.
For the ongoing collaboration with the PEI we modified our VLPs.

Week 39

Finally got some good results after having another go at doing a Western-Blot of our proteins.
To insert mCherry with a TVMV-cleavage-site into pDESTara2, we switched to Gibson Assembly.

Week 40

Finished cloning sfGFP and Sortase A7M into pDESTara2.
We did a solid phase protein synthesis (SPPS) of a polyG-peptide for a Sortase A7M reaction to inspect turnover and steric hindrance of this reaction.
We also started doing FRET-assays of mCherry with GFP. Unfortunately, this didn’t work, as the spectrals did not overlap enough for our purposes. Consequently we conducted a FRET-assay using mCherry and TAMRA, which gave us the expected results. However TAMRA bleached our samples too much, in fact more than the actual FRET-effect did. Finally, when we switched to using sfGFP as the FRET-donor and TAMRA as the FRET-acceptor, we were able to start testing different ratios.
As the assays using copper-click-reaction didn’t work, we conducted a mass spectrometry of the primary amines linked via Sortase A7M to TAMRA-LPETGG. This showed the functionality of the sortase.
Besides producing VLPs in vivo and purifying them, we also inspected them in a SDS-PAGE, which confirmed that our VLPs can be modified.

Week 41

We finished the cloning of mCherry and the TVMV-protease-cleavage-site into pDESTara2.
We conducted a dual expression assay of sfGFP and mCherry in pTeTW3con2.
Protein purification of FLIPR-TEV-LPETGG. Our VLPs, assembled in vitro, were modified using FLIPR-TEV-LPETGG for our collaboration with iGEM Freiburg.

Week 42

The modified VLPs, using FLIPR-TEV-LPETGG, were sent to the iGEM Team Freiburg for further use in their project.
We also conducted more assays with Sortase A7M and Sortase A5M and further FRET-assays.

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