Difference between revisions of "Team:Amsterdam/LabJournal"

Robustness analysis. Take optimal flux of the shunt and start decreasing it, so now your mu is going to drop. reduced cost analysis. Conflicting goals of iGEM and Joeri’s project. For iGEM it is necessary to do shorter experiments with more replicates. Evolution is not interesting for iGEM. Fluctuations part might be important for iGEM. First constant light, then smooth di...regime. An easy way to increase fumarate production. Start with omics data, because this feeds into the other modules.

ML vs ICL and ML: test if it is thermodynamically possible to make this reversible. You have clear comparison if it reversible. This should be done in the targeted module. The synthetic biology part in E. coli is similar for both projects, so we need to team up: MS by Bram and ICL & MS by Yuki. We have to consider different types of MS (E. coli, codon optimized from E. coli). The physiology part in Synechocystis is totally different for both modules. Bram uses known promoters, Yuki uses promoter library. The analyzation of the timing of the shunt should be done by Bram with the known promoters. Additional experiment: Get whole library (this is a mix), apply regime, grow until OD. The ones that are still in there perform best.

You cannot put kinetics into genome model. You can make tables with affinity (Km and Kcat) and make your choice based on this. Take the maximal predictive flux and see whether this is compatible with the affinity of the transporter. You have to make a rational choice.

Linker length is really important. On paper it is quite easy, but in the lab it might be hard. If all else fails, and this works we could present this alone and it will still be good. Calculate CO2 → fumarate efficiency (E. coli vs Synechocystis). From biosensor track to transporter track. Maybe ask a econometrist to do this part. Add a sensitivity analysis.

Title and project description. We have to think of a title. We have to be careful for the political correctness of the presentation and title. We have to think about the standards of the other countries. Examples: “Making fumarate around the clock.” “Sustainability day and night. “ Biosafety Strains all always outperformed by wildtype. They cannot take over the wildtype, so it is contained. Even if there is contamination, you do not put marked genes in, because our genes are markerless.

• Max made a model using FAME software.
Next:
• find optimal growth rate and fumarate production for MS flux.
• look into growing on glycogen instead of glucose.

• We use biobricks to fuse the two genes together
• Orientation of TA cloning: by Colony PCR primer design.

• Design plan for the lab with Wei.
• Found one potential C4-dicarboxylate transporter in Synechocystis
• Set up pipeline
• Discussed multiple transporter families in bacteria.

• We found that previous systems did not use linkers, so we will also not do this. We will amplify dcuS from the E. coli genome with different lengths, however, both TM domains will always come from dcuS and the kinase will always entirely be envZ.
• Do parallel experiment: …..
• Age is designing primers now. Wei proposed to take different lengths how many different lengths to take from dcuS and how to decide different cutoffs (how much difference should there be)?
• How can you choose the direction of your gene on the T-vector?
• In Wei’s protocol it says ecoRI is in all T-vectors, but snapgene does not identify them as unique restriction sites (they are there but are not unique, does this matter?)
Two research questions:
• How is fumarate transported in Synechocystis?
• How can we increase fumarate transportation?

• Experiment to test the software, starting Friday over the weekend.
• Write up on the graphs from last time, it is on the drive: location.

Targeted approach:
• Bram and Yuki made solutions in the lab with Wenyang.
• Bram failed his first experiment.
• Bram and Yuki designed primers for the zwf knockout. We both have a file on the drive. The primers from the file from Yuki are already ordered by Dennis and will be used for the markerless fusion-pcr: slr1843-zwf_Yuki .

Untargeted approach:
• Made a plan for the untargeted incorporation of the glyoxylate shunt: plan_yuki (if you download it you can zoom in to look at the details)
• Made an account on Benchlink, to access all the files
• The first step will be the transformation of mazF-Sp into ∆fumC as the receptor species.
• Isocitrate lyase (ICL) and malate synthase (MS) need to be fused, with two different rbs in front of the ICL and MS. Patricia only uses cpc, so we need to find another rbs sequence from literature (Englund et al., 2016).
• We need controls to make sure both rbsses are working
• Then ICL-MS needs to be incorporated into a t-vector. Therefore, the restriction sites in ICL-MS need to be checked. Then, we can design primers to fuse ICL and MS and incorporate in into the t-vector.

So the next steps for the untargeted approach will be:
• Making solutions (LB and BG-11), plates, antibiotic stock etc. - together with others?
• Deciding what rbs to use from literature
• Check restriction sites in ICL-MS and possibilities for the t-vector.
• Design primers for fusion PCR of ICL-MS.

New pipeline:

New appointment with Anton

• Designed primers for the biosensor, there has been a slight change in plan -> the Acc65I restriction site on dcuS has to be mutated first
• Made a multiple sequence alignment of dcuS, envZ and cph1, looks well aligned (TMs, HAMPs and HK’s align), might be interesting to look at this with someone with more expertise.
• Discussed possibilities for an intracellular sensor with Klaas (FRET) -> he will ask a connection about a possible fumarate binding protein for this system

Exam Week

Exam Week

Exam Week

• Started pilot experiment on Monday: 7 of 8 vessels are doing well, the 8th not so much
• The 7 are currently in photonfluxostat mode but I will switch them over to the sinusoidal regime later today
• I keep getting contamination when I plate samples on LB plates. However, I don’t think this is due to the vessels being contaminated but rather it has to do with my sampling/plating technique because 1) the contaminant colonies are usually not where I pipetted samples and 2) contaminates have “disappeared” from vessels after multiple platings
• Worked out how much 15N we will need: (less than a gram). I think it was ordered yesterday
• The dFBA model

Targeted approach
• Learning techniques in the lab. Communication with WenYang goes well.
• PCR - amplified H1 and H2 of zwf region. Fused H1 and H2.
Untargeted approach
• Made solutions (LB, BG-11) and selection plates.
• Revived the Synechocystis ∆fumC and wildtype strain. They are on BG-11 plates and ready for transformation.
• Revived the E. coli pFLNX H1H2 MazF Ω 1.1 #5.
• Isolated the MazF plasmid and stored it for transformation into ∆fumC.
• Transformed the plasmids that we received from other labs, including the pAQ MS-ICL with the glyoxylate shunt enzymes isocytrate lyase (ICL) and malate synthase (MS). Inoculated in and this afternoon we will perform miniprep to isolate the plasmids.
• Working on a new approach to make the plasmid below:

Selected genes and project overview on drive.

Josine needs conclusions on which ones to use and which ones not to use. Make a meeting where you present what you found so far and which transporters specifically you want to express in which strains. Critically go through what you found. Josine has to convince us.
You are planning to overexpress this is in Synechocystis as a final product. First, knock this promoter out to see if it has an effect on fumarate transportation. See what the impact is.

• Made the overview, working in the lab

• Met with Henk the other day to talk about proteomics prep procedures, 15N is on its way. Monday they will have meeting with Henk.
• Joeri and I have a meeting with Martijs Jonker and Rob Dekker to discuss transcriptomics on Monday at 15.30. Filipe wants to be there.

Targeted approach:
• Constructing the zwf knockout plasmid
• Develop plan to make the 36 constructs as parallel as possible
Untargeted approach:
• Transformed the selection cassette MazF-Spec into the fumC-knockout.
• New approach to get the glyoxylate shunt enzymes into the H1 H2 plasmid.
• Check if the unannotated region contains a promoter or not
• BLAST search
• Promoter prediction

• Primers for knocking out Sll1103 and Sll1314 are designed, checked and will be ordered. (Both TRAP transport-domains)
• Bram will explain me the pipeline, Wei will guide me with the photoheterotrophic condition experiment, but I need supervision to start in the lab.
• First, Parsa will help Josine for the first 1,5 month. Then Davide will come in to help.

• Hopefully mutated the dcuS gene.
• pCI148.

• Working with Henk tomorrow. Testing out the proteomics procedures.
• Carbon efficiency calculation

Targeted
• Zwf-KO plasmid is at sequencing
• Made a scheme to start with constructing the plasmid of the integration of the promoter system.
Untargeted
• Confirmed plan with Patricia and Wei: started yesterday in the lab.
• Growth rate will be higher, select for this. Fumarate is aligned with mu. You select on mu.
• Library of e. Coli promoters into synechocystis.

• Started in the lab → amplifying homologous regions up and downstream of the gene. Knock-out will be markerless.
• Making a wet-lab scheme to follow the progress

Amplified all 9 different dcuS parts successfully, working on envZ half now.

Exam Week

Exam Week

Exam Week

Exam Week

All single knockouts

R_RBCh: 1 O2 1 D-Ribulose 1,5-bisphosphate ==> 2 H+ 1 3-Phospho-D-glycerate 1 2-Phosphoglycolate
R_PGLYCP: 1 H2O 1 2-Phosphoglycolate ==> 1 Phosphate 1 Glycolate

R_DDPGA - 1 4-Hydroxy-2-oxoglutarate <=> 1 Pyruvate 1 Glyoxylate
R_EHGLAT - 1 2-Oxoglutarate 1 L-erythro-4-Hydroxyglutamate ==> 1 L-Glutamate 1 4-Hydroxy-2-oxoglutarate
R_PROAKGOX1r - 1 O2 1 L-Proline 1 2-Oxoglutarate ==> 1 Succinate 1 CO2 1 trans-4-Hydroxy-L-proline
R_PHCD - 1 Nicotinamide adenine dinucleotide 2 H2O 1 L-1-Pyrroline-3-hydroxy-5-carboxylate ==> 1 H+ 1 Nicotinamide adenine dinucleotide - reduced 1 L-erythro-4-Hydroxyglutamate

Targeted
• Still need to work on the detailed plan of Wei
• Setback in PPP knockout. Used wron T-vector
• WenYang never did experiments as we have planned with the CPH8 system

Untargeted
• Making a detailed schedule with Patricia, including non-lab work to speed things up and get a clear overview
• Again transformation of MazF cassette into ∆fumC

H1-H2 fused for both constructs (sll1103 and sll1314), placed in the T-vector (ligated). Will start transformation procedure next week.

Experiments in ~2 weeks, thinking about experimental conditions (too much now): Strains:
• pCIdcuSZ 1-9 + pCI146 (9×)
• pCIdcuSZ 1-9 (9×)
• pCI146
• WT
• pCI148?
• pCI148 + pCI146?
Fumarate in mM:
• 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10
Compounds:
• Fumarate, succinate, malate, aspartate
Time points:
• 0, 2, 4, 6, 8
Other:
• Continuous light vs. dark
• LB vs BG11?

• Started dummy 15N experiment
• Struggling with modelling protein synthesis/degradation with FBA in a meaningful way
• Theoretically able to do the entire proteomics procedure by myself. Henk will be back next week and then I will test that theory
Possible ways to incorporate proteomics data into the FBA model
• proteomics defines upper constraints
• Colijn, C., Brandes, A., Zucker, J., Lun, D. S., Weiner, B., Farhat, M. R., … Galagan, J. E. (2009). Interpreting expression data with metabolic flux models: Predicting Mycobacterium tuberculosis mycolic acid production. PLoS Computational Biology, 5(8).
• proteomics defines objective function, may be useful during night
• Montezano, D., Meek, L., Gupta, R., Bermudez, L. E., & Bermudez, J. C. M. (2015). Flux balance analysis with objective function defined by proteomics data-metabolism of mycobacterium tuberculosis exposed to mefloquine. PLoS ONE, 10(7), 1–19.

Targeted
• Performing two approaches. Sending slow 1 for sequencing this afternoon. Sending fast one tomorrow.
Untargeted
• Waiting for sequencing primers for the pFLXN-H2-rbs-MS-rbs-ICL-Kan-H1 to arrive before I can sequence it.
• The transformation of the mazF omega cassette into ∆fumC did work, contrary to what Patricia and I thought (yellow dots). Colonies have been picked and regrown to check: no contamination. Colony PCR on these colonies shows that the colonies contain the mazF omega cassette.
• Look into Electroporation for transformation.
• Primers for segregation have to be designed: Send sequence to Wei to see if he already has them.
• Transform ligation mixture again today to check new colonies tomorrow.

Miraculously took the undigested T-vector instead of the digested one: explains why I got the wrong colony PCR bands and why it did grow on Amp-plates. Started over (I threw away the wrong eppies). Learned a lot.

• Double digestion of all 9 dcuSZ fragments and the pCI148 plasmid with Acc65I and MunI failed. Seperate digestion (after pcr purification) to check seem to show that the enzyme is working really slowly, tried digestion with only Acc65I overnight, this failed -> i probably screwed up the miniprep so the plasmids are not cuttable.
• Got some sequencing results back: what is a good/efficient way to check these?: LaserGene (was DNA Star)

15N trial run is still in the first (pre-culture) phase
• analyzed fruits results
• degradation rate on just malate synthase

Targeted
• -
Untargeted
• Waiting on sequencing primers for my construct
• learned that these things can unexpectedly take a lot of time - think ahead
• Extracted genomic DNA for the first time: the ∆fumC colonies with hopefully the mazF Ω cassette.

Back to where I was, now with full back-up stocks of DNA, constructs, everything PCRred to the max.
Will do ligation today.

Things are going slower than expected, ligation failed and I am out of pCI148. Going to do maxiprep today to have enough pCI148 (low copy number), I will digest the plasmid and do ligation over the weekend. I still have the dcuSZ1-9 fragments.

Exam Week

Exam Week

Exam Week

Exam Week

• almost done with the light fluctuating protocol
• started the real 15N pre-culture
• trial 15N is in the second phase (11 different culture conditions)
• how exactly should I go about harvesting/mixing?
• working through proteomics protocol mostly on my own
• I need to take a step back from some of my iGEM responsibilities so I can write my grant proposal and pro

Targeted
• Transformed zwf-h1-h2 to Synechocystis.
• Ordered Cph8 construct partly gene, partly gblock
• Transformed WT and delta FumC strain WTmazf strain
• Waiting to transform and FumCmazf untill Yuki has it finished: it is finished probably tomorrow
Untargeted
• Sequencing results MS-ICL look good, but:
• one primer annealed in a different place than designed - sequence this part again
• ∆fumC mazF Ω transformation - a lot of problems amplifying the fragment
• reinoculated into twice as much antibiotics - ready tomorrow
• gDNA extraction protocol for high scale: high concentrations, also after cleaning with RNAse (clean on gel)
working on improving protocols with Patricia
• partial digestion (BglII, BamHI, Sau3AI)
• transformation by electroporation

• Sequencing results look good
• sll1103

• sll1314

• MazF cassette ligated → transformed → colony PCR: confirmed right construct.
• Monday start with transformation to Synechocystis (now preparing strains and media).
Start new project
• Primers designed for the overexpression of DcuB and DcuC from E.coli
• Designed gene for synthesis (DcuC from N. Spumigena, 1.300 bp)

• I am very carefully and slowly reperforming the ligation that failed twice
Sequencing results of the T-vector containing the show one amino acid mutation:
• Valine -> Isoleucine
• 25th a.a. in dcuS (TM1)
• Similar a.a. both hydrophobic; Blast search shows 1 dcuS sequence that has this same mutation (100% similarity with sequencing result)

• fluctuation protocol is finished (for now)
• current proteomics protocol is only picking up ~200 proteins
• will talk to henk about using the protocol in:
• Borirak, O., de Koning, L. J., van der Woude, A. D., Hoefsloot, H. C. J., Dekker, H. L., Roseboom, W., … Hellingwerf, K. J. (2015). Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803. Biotechnology for Biofuels, 8(1), 111. https://doi.org/10.1186/s13068-015-0294-z
• and/or may try hilic chromatography
• 15N pre-culture is almost ready (OD: ~1.4), will most likely start the 2nd stage over the weekend

Targeted
• Transformed zwf-Mazf to FUMC-OmegaMazf and also to WT-Mazf-Omega.

• Re-inoculate deltaFumC-zwf and WT-Omega
• I was not able to digest the vector pFLXN with 4 enzymes. After 3 tries I found that the NedI enzyme is not cutting the plasmid. Since it is FLXN it should contain it. So now checking if a new enzyme stock works.
• I go on holiday this saturday. I arranged a planning with Wen Yang to continue the steps in Synechocystis.

Untargeted
• ∆fumC mazFΩ is ready: glycerol stocks in -80C
• Get also evidence without marker
• Planned to digest gDNA with Sau3AI and MS-ICL with BglII this week and ligate over the weekend -but Sau3AI is not in yet..
• Missed part for sequencing, send again - no results yet
• Making competent cells from different protocols to test efficiency: iGEM Kit Competent Cells test (-20C). Made an excel file that calculates the efficiency from the amount of colonies using this kit.
Tip to check concentration:
• Load 5 uL ladder, 1 uL sample. You would at least have the same concentration of your band and your sample, because then it is five times higher than your ladder (compare bands). Filipe says your concentration must be 100.

Knock-out-project
• Transformation postponed constantly..
Overexpression project
• Primers ordered for the overexpression of DcuB and DcuC from E.coli
• Gene sent for synthesis (DcuC from N. Spumigena, 1.300 bp)

• Fully confirmed 7 of the 9 constructs by sequencing, missed a part for construct 3 and construct 6, send those parts for sequencing again
• Ligation is still not working; using Wei’s approach instead of Parsa’s now (less DNA in less volume instead of more of both) -> also checked previous ligation with highly competent cells from Mara, no result
• Inserts seem good when checked on a gel
• Only doubt could be pCI148 (there is an undefined smear on the gel) and volume size of ligation mixture
• Redigested pCI148, checked on gel: very low concentration, can’t see an undefined smear now (can also be because of low concentration)
• I will try to ligate this to one of the inserts in different ratios (used 1:3 up till now), extracting more pCI148 next week.

2nd stage of 15N is underway. Harvested the “non-growing” cultures yesterday

Targeted
• I also tried to digest the pFL-XN plasmid containing H1H2 with four enzymes and found just like bram that NdeI did not cut. Inspection of my own sequencing results (on pFL-AN) showed that the NdeI site is actually not on the plasmid.
• Continued with the two fragments, blunted ends, checked on gel → unexpected bands showed up after blunting?
• Extracted correct bands from gel, I will start ligation tomorrow so bram can do transformation when he comes back.
• For ligation: is the formation of ori-ampR-ori-ampR (instead of ori-ampR-H1H2) plasmids a big problem? (everything is in one mix)
Untargeted
• Confirmed the sequence of pFLXN-h1h2-rbs-MS-rbs-ICL
• primer that was designed to amplify the BglII region (where my promoters will be inserted) annealed again at a total random place - almost no match in sequence.
• Doing a lot of preparational work - Patricia is going away for a couple of weeks
Testing protocols to digest genomic DNA with Sau3AI:
• overnight incubation (~16 hours)
• 20 µg DNA with 3 µl enzyme
• 10 µg DNA with 2 µl enzyme
• Question from Patricia: can we order the large tube with Sau3AI? The tube we received only has enough for 8 digestions - maybe order before Dennis goes away.
• Designed new primers to amplify the mazFΩ fragment in ∆fumC.
• Ligating the gDNA into pFLXN h1h2-rbs-MS-rbs-ICL over the weekend.

Project knock-out
• Transformation to Synechocystis → now in the stove on BG11 plate (with Kanamycin)
Project overexpress
• Amplified DcuC
• Amplified DcuB (Fusion PCR for DcuB to mutate NdeI in the gene)
• Ligation today over the weekend.
• Next week transformation, colony PCR and sequencing check.
• Have done all of this before already, so do not need supervision coming week.

• All constructs fully confirmed by sequencing
• Checked if heat inactivation of enzymes makes a difference after digestion of pCI148; answer is no it does not seem to (at least on gel)
• Checking if the pCI148 plasmid can be fully digested, this has not yet succeeded
• Anniek told me plasmids of this origin are very annoying because they are naturally prepared for mobilization, this means they are often single stranded and folded in the organism, when you extract the single stranded plasmid it cannot be cut by restriction enzymes, this can often lead to ‘false positives’ in your ligation
• Photanol does not use this plasmid anymore but Anniek still has a strain which contains a mutated version of the RSF1010 plasmid which does not get prepared for mobilization (from research by golden lab), maybe i could take this plasmid, cut everything i need out of my plasmid and put it in this one?

• Leo is currently running an old sample on a 2 hour gradient instead of the previously used 30 minute
• Will also run samples using hilic separation this afternoon
• Centrifuging works
• Starting turbidostats next week

Targeted
Ecoli.
• pFL-XN-H1H2-0168 without restriction sites is constructed and is send for sequencing to check the direction of the ligation.
• IDT sequences arrived, picked them up this morning. I ordered my sequence in three parts. I will dilute them and try to clone the three parts together. (With Wei if possible)
Synechocystis.
• Waiting for full segregation of WT-zwf
• Waiting for full segregation of deltaFUMC-zwf-Mazf
• WT-MazF-zwf and Fumc-zwf are not growing, so maybe try once more at perfect OD for the third time. (With Wei if possible)

Untargeted
• Started segregation experiment for Synechocystis with Patricia - plate everyday (Wednesday, Thursday, Friday, Saturday) and then every other day (Monday, Wednesday etc).
• I will make a plan with Patricia for when she is on a holiday to continue some running experiments.
• Digested the gDNA and my plasmid with the glyoxylate shunt genes and ligated together. Going to transform today.
• Made competent cells from Mara's protocol (XL1Blue). Tested efficiency - looked online for heat shock protocol.
• 90 sec is standard in the lab
• 45 sec was recommended
• >> better efficiency with 45 sec heat shock
• gDNA extraction: 10 min vortex vs 10 min beadbeater
• Testing now
• reinoculated ∆fumC mazFΩ into BG-11 without antibiotics to check segregation - how long should this grow?