Team:UC Chile/Lab NoteBook

From 2013.igem.org

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Latest revision as of 23:11, 26 September 2013

Wiki-IGEM

Lab Notebook

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We didn't work that day.
Summary of Week (1-7)
We are still working on getting used to work in a Biology lab. For some of us it is the first time doing wet lab work.
Summary of Week (8-14)
We mostly worked on our Agro project, focusing on characterizing GV3101 strain, pCAMBIA (our control for binary vector) and getting useful biobricks out of the 2012 iGEM distribution kit.
Summary of Week (15-21)
pHnCBS1D was sent to us! Thanks a lot ---- ! We started working on our Carbo project by making our own stock of this important plasmid. Also, we took some of the biobricks from the 2012 distribution kit necessary for Carbo. We digested and corroborated the Agro’s 2012 biobricks.
Summary of Week (22-28)
We focused on making the constructs and experiments for the INGENIA fair (one of our Human Practices). Since we know it takes them a bit of time to grow, we started planting Nicotiana and Arabidopsis seeds. At the end of the week we received Carbo’s primers!
Summary of Week (29-5)
We prepared the parts for the Gibson Assembly of C.SG and C.GS by doing lots of PCRs but we are having problems amplifying the backbone. Are our primers forming heterodimers?
Summary of Week (6-12)
AddGene toolkit for constructing TALENs is with us now! We worked on getting all the necessary plasmids for the specific TALEN sequence we want to build. We also tried different strategies for the amplification of the Carbo’s backbones (such as: Hot start touchdown PCR, changing the buffer for one with GC+DMSO, changing the template, etc). After a lot of struggle we solved the problem . At the end of the week we received Agro’s primers!
Summary of Week (13-19)
Finally, with all our parts for the C.SG and C.GS strategies we worked on Gibson Assembly and E. coli transformation of competent cells. Also, tried out for the first time Golden Gate Assembly for building the TALENs, however, we didn’t get any correct results.
Summary of Week (20-26)
We have our first construct: C.SG ready ! We are sooo happy! C.GS is showing unexpected results for the colony PCR and the digestion. We are going to try it all over again. Also, we found out that our reagents XGAL and IPTG were not working correctly  so, lots of positives and negatives controls were done in order to check for more mistakes like this one.
Summary of Week (27-2)
Golden Gate Assembly didn’t work this week either. We got the primers for the C.LG and C.GL strategies so we immediately tried to do Gibson PCR for the two of them and the C.GS construct. We also started working on generating a vector for the knockout of the VirD2 gene in Agrobacterium, but didn’t get what we expected.
Summary of Week (3-9)
We are still trying to get C.LG, C.GL and C.GS to work with little success. We also cannot find the reason why the Golden Gate Assembly: Day 1 is not functioning properly. GFP activity on Nicotiana and Arabidopsis was measured using microscopy. Co transformation of pHnCBS1D and C.SG is progressing smoothly.
Summary of Week (10-16)
Induction of co-transformation of pHnCBS1D and C.SG is giving us pretty good results. We found out lots of mistakes we were making, we fixed them and got C.LG and C.GS constructs working. Golden Gate Assembly is still not working.
Summary of Week (17-23)
Induction of co-transformation of pHnCBS1D with both C.LG and C.GS showed GFP activity under the microscope! Also, our team successfully made the Homologous Recombination vector. The presence of GFP in Nicotiana’s genome was corroborated by PCR.
Summary of Week (24-30)
We mostly focused on working with our C.RFP strategy. We actually had to order some extra primers to amplify the whole backbone. C.GL is still giving us some troubles. We got everything settle to transform Agrobacterium and knockout VirD2.
April 30th
  • Run gel with Gibson PCR for C.SG and C.GS strategy.Only GFP C.SG, C.SG and C.GS promoters worked.
  • Cut gel of: C.SG promoter, C.GS promoter and C.SG GFP.
  • Do Gibson PCR of C.SG and C.GS parts that didn’t work but changing PCR cycling conditions.
April 29th
  • Run gel with Gibson PCR for C.SG and C.GS strategy. Didn’t work at all.
  • Do Gibson PCR for C.SG and C.GS strategy again.
April 26th
  • Make new 1k ladder.
  • Run gel with digestion of: Constitutive promoter BBa_J61002 and Linker of 15aa BBa_K157000. They both appeared to work.
  • PRIMERS are here!!!.
  • Make MM2X HF for Gibson PCR.
  • Do Gibson PCR for C.SG and C.GS strategy.
April 25th
  • Sterilize Arabidopsis GFP seeds.
  • Digestion of:Constitutive promoter BBa_J61002 and Linker of 15aa BBa_K157000.
April 24th
  • Work on primer design.
April 23th
  • INGENIA fair.
  • Make media for planting Nicotiana GFP and Arabidopsis GFP.
  • Sterilize Nicotiana GFP seeds.
April 22th
  • Check out E. coli transformation with the INGENIA fair constructs.
  • Make plates with LB + Km (x4) and LB + Cm.
  • Make drawings and interesting bacteria stuff for the INGENIA fair.
April 19th
  • E. coli transformation with the INGENIA fair constructs.
  • Miniprep of: pSB4K5 (backbone), Constitutive promoter BBa_J61002, Linker of 15aa BBa_K157000 and pHnBCS1D.
April 18th
  • Make liquid cultures of pSB4K5 (backbone) and Constitutive promoter BBa_J61002.
  • Make plates with LB+Km (x10) and LB+Cm (x1).
  • Prepare constructs for the INGENIA fair.
April 17th
  • Liquid cultures of E. coli transformation with Linker of 15aa BBa_K157000.
  • Liquid cultures of E. coli transformation with pHnBCS1D.
  • Make plates LB+Km.
  • Take out of the 2012 distribution kit: backbone pSB4K5 (plate 1 -Well 5G), Constitutive promoter BBa_J61002.
  • E. coli transformation with pSB4K5 (backbone).
  • E. coli transformation with Constitutive promoter BBa_J61002.
  • Make SOC.
April 16th
  • E. coli transformation with Linker of 15aa BBa_K157000.
  • Take out of the 2012 distribution kit: Linker of 15aa BBa_K157000.
  • Run gel with digestion of pOR1. pOR1 is OK.
  • E. coli transformation with pHnCBS1D (carboxisome’s plasmid).
April 15th
  • Digestion of pOR1.
  • Work on primer design.
April 12th
  • Miniprep of pOR1 (binary vector).
April 11th
  • Liquid cultures of saved pOR1 (binary vector).
  • Made a list of all reagents we are going to need for our iGEM project.
April 10th
  • Plates of LB+Km were not done correctly :O. Tried to save pOR1 (binary vector).
April 9th
  • Isolation of pTI from Agrobacterium tumefaciens (strain from another lab).
  • 25 mL of Glucosa 1M.
  • Make SOC.
  • Make plates LB+Km (x6).
  • Take out of the 2012 distribution kit: pOR1 (binary vector).
  • E. coli transformation with pOR1 (binary vector).
  • Meeting with the boss.
April 8th
  • Growth curve of Agrobacterium tumefaciens (strain from another lab).
  • Digestion of pCAMBIA.
April 5th
  • Miniprep of pCAMBIA.
April 4th
  • E. coli transformation with pCAMBIA worked. Grew too much so, we had to re-streak a single colony.
April 3rd
  • Make SOB.
  • Make SOC.
  • Make plates with LB + Km (x6).
  • E. coli transformation with pCAMBIA 1301.
May 31st
  • E. coli transformation with Golden Gate Assembly (pFUS_A).
  • Plates with C.GS, C.LG, C.GL and Homologous Recombination are all wrong!.
  • Make Gibson Assembly of C.GS, C.LG, C.GL and Homologous Recombination again.
  • Plates LB + Km (x6).
  • Send C.SG for sequencing
  • Check root of Arabidopsis for GFP. → Nothing → Cry
May 30th
  • Run gel of Gibson PCR of C.LG, C.GL and C.GS. Everything worked except for (backbone+promoter)..
  • Gel purification of bands with the expected sizes.
  • Microscopy of Nicotiana to check for constitutive expression of GFP. Couldn’t see much. We need to check if maybe it’s expressing GFP in a particular tissue
  • Make plates with LB + Km + Cm (x4).
  • Golden Gate Day 1 for pFUS_A again (with new extracted plasmids).
  • Gibson Assembly of C.GS, C.LG, C.GL and Homologous Recombination.
  • E. coli transformation with C.GS, C.LG, C.GL and Homologous Recombination.
May 29th
  • Run gel with Gibson PCR for Homologous Recombination.
  • Miniprep of C.SG.
  • Golden Gate Assembly Day 1 again.
  • Digestion of some of the new minipreps of plasmids from the AddGene kit.
  • Gibson PCR of C.LG, C.GL and C.GS again (this time using C.SG as template).
May 28th
  • Do Gibson PCR to generate the plasmid for Homologous Recombination in Agrobacterium tumefaciens.
  • Run gel with the Gibson PCR of plasmid for Homologous Recombination . →We didn't get anything→ Cry
  • Make liquid cultures of C.SG.
  • Do Gibson PCR of the parts for C.LG and C.GL strategies.
  • Do Gibson PCR of C.GS backbone again.
  • Plates with LB + Km (x8).
May 27th
  • Made Gibson PCR of all the parts for C.GS again (same procedure as before).
  • Run gel with Gibson PCR of C.GS.
  • Primers for C.LG and C.GL are here!.
May 25th
  • Check out positive control for the XGAL and IPTG. →XGAL is working!
May 24th
  • Tried digestion of pSB4K5 and C.SG and Didn’t work.Probably a restriction enzyme was too old.
  • Digestion of pSBK45 and C.SG. Beautiful bands for both! We have our first construct C.SG ready :).
  • Make new plates LB + XGAL+ IPTG + Spec and plate another positive control.
  • E. coli transformation with C.GS didn’t work again.
  • Organize the lab and throw old stuff .
May 23rd
  • Miniprep of pSB4K5.
  • Miniprep of C.SG.
  • Positive control for the XGAL and the IPTG only showed white colonies :0. Make new stock of XGAL and IPTG.
  • E. coli transformation with C.GS didn’t work again.
  • E. coli transformation of C.GS again (to check if it was a problem of the transformation of competent cells or the Gibson Assembly itself).
May 22nd
  • Plant Nicotina (from plates to pots).
  • Make plates with LB + XGAL + IPTG + Spec.
  • Make a positive control for the XGAL and the IPTG (to check that the reagents are working).
  • Liquid cultures of pSB4K5 (Pbad promoter).
  • Liquid cultures of C.SG.
  • Colony PCR of C.SG . We got the expected sizes!!<
May 17th
  • Stock of Spectinomycin 50mg/mL.
  • Miniprep of TALEN plasmids.
  • Run gel with the colony PCR of C.GS.→Didn’t get the expected sizes→Cry
  • Run gel with the Golden Gate Assembly. Ugly gel, really blurry, couldn’t read the sizes of the bands properly.
May 16th
  • Make LB.
  • Make MM 2X HF.
  • Golden Gate Assembly Day 1 for pFUS_A.
  • Miniprep of p_FUSB6.
  • Liquid cultures of single colonies from the C.GS transformation.
  • Colony PCR for C.GS.
  • E. coli transformation of C.SG again.
  • Liquid cultures of required plasmids from the AddGene kit again. (we had too little concentration)
May 15th
  • Golden Gate Assembly Day 2 (colony PCR for the detection of positive assemblies and liquid cultures). pFUS_B6 seems to be ok. pFUS_A didn’t show good results.
  • E. coli transformation with pSBK5 (with Pbad promoter).
  • Plates with LB + Amp + IPTG + XGAL (X4).
  • E. coli transformation with the Gibson Assembly of C.SG and C.GS. Weird white precipitate was seen after 1 hour incubation of the cells.
May 14th
  • E. coli transformation with the Golden Gate Assembly.
  • Run gel with GFP C.SG and promoter for both strategies. Gel was ok.
  • Cut bands.
  • Run gel with the digestion of TALEN plasmids. Plasmids with the Tet resistance seem to be ok. pFUS_A looks a bit weird (maybe because of a partial digestion?)
  • Gel purification of all the parts for the C.SG and C.GS construct.
  • Measure DNA concentration of the parts.
  • Run gel to check that the purification was ok. All the bands were ok
  • Make MM for the Gibson Assembly.
  • Gibson Assembly of C.SG and C.GS.
May 13rd
  • Golden Gate Assembly Day 1.
  • Digestion of TALEN’s plasmids. Concentrations were too low so we had to concentrate them first.
May 11st
  • Miniprep of pNN7 and pNN2.
  • Agro’s primers are HERE!!!.
  • Nanodrop of TALEN’s plasmids.
May 10th
  • New liquid cultures of pNN7 and pNN2 because they didn’t grow again.
  • Miniprep of the plasmids from the TALENs kit.
  • Plant out Arabidopsis (from media to pot).
  • Change Nicotiana to new plates.
  • Run Gibson PCR of: Rubisco Small C.GS, GFP C.GS, GFP C.SG again (the whole volume this time).
  • Cut gel of the correct part.
May 9th
  • Run gel with Hot Start Touchdown PCR for the amplification of the backbones.→ Didn't work → Cry
  • Do another Gibson PCR (positive control, prefix – sGFP, sGFP – suffix and Rubisco Small).
  • Runn gel of Gibson PCR. Worked perfectly, problem with the backbone solved :).
  • Cut bands.
  • Re –streak of pNG1, pNI3, pNI7, pNI4, pNN7, pNN2, pNN10, pNN8, pHD4, pHD8, pFUS_A, pLR_HD and pTAL_3 because plates got dry. The other plates were saved in the cold chamber.
  • New liquid cultures of the plasmids that didn’t grow: pNNI3, pNG1, pHD6, pNN8, pNN7, pNI4, pNN2 and pNN5.
May 8th
  • Liquid cultures of plasmids from the AddGene kit.
  • New strategy for the amplification of C.SG and C.GS backbones: PCR amplification of [backbone + GFP] from another template (last year’s iGEM group had one). New cycling conditions: Hot start Touchdown PCR..
May 7th
  • Buy dry ice.
  • Make plates with LB + Tet (x 21), LB + Spec (x 4) and LB + Amp (x2).
  • Plate in a Petri dish the required plasmids from AddGene TALENs Kit.
  • Touchdown PCR from original backbone again (to amplify C.SG and C.GS backbones).→ Didn't work → Cry
May 6th
  • Check out the sequences for the TALENs (to know which plasmids we need from the kit).
  • Run gel with May 5th touchdown PCR.→ Nothing → Cry
May 3th
  • Run gel with the band stab PCR and touchdown PCR. We got GFP and Rubisco Small for the C.GS strategy. Touchdown PCR worked but the band is too weak.
  • Digestion of pHnBCS1D . One of our minipreps gave us the correct result. The other one was discarded .
  • CMake MM2X GC.
  • Make MM2X GC + DMSO.
  • Band stab of C.SG and C.GS backbones and touchdown PCR again..
May 2th
  • Run gel with Gibson PCR of the parts that didn’t work on April 30th. RubisCO small for the C.SG seems to have worked.
  • Cut gel of RubisCO Small of C.SG strategy.
  • The band for GFP of C.GS strategy was too weak. Band stab and Gibson PCR were done again.
  • Make MM2X GC.
  • Make MM2X GC + DMSO.
  • Try to figure out why the other Gibson PCRs are not working. We think backbone’s primers are forming heterodimers! :0.
  • Try new strategy for C.SG and C.GS backbones: 1. Change Master mix to MM2X GC and MM2X GC + DMSO 2. Change cycling conditions: Touchdown PCR..
June 3rd
  • E. coli transformation with pHnCBS1D.
  • Check out C.GL, C.GS, C.LG and Homologous Recombination vector. We didn’t get any transformants.
  • E. coli co-transformation with pHnCBS1D and C.SG.
  • Golden Gate Assembly: Day 2 of TALENs (pFUS_A).
  • Golden Gate Assembly: Day 1 of pFUS_B5.
  • E. coli transformation of pFUS_B5. Because of an error on the planification for the assembly of the TALENs we were mistakenly working with the array pFUS_B6 when we should have done it with pFUS_B5,.
June 4th
  • Send C. SG for sequencing. :).
  • Gibson PCR of C.LG, C.GS, C.GL (not using C.SG as template).
  • Gibson PCR of Homologous Recombination vector.
  • Plating from the Addgene’s kit: pFUS_B5 (for the Arabidopsis’s TALENs) and the modules, arrays and last-repeat for the Nicotiana’s TALENs . This means plating of pHD2, pNG2, pNG3, pNG4, pNG5, pNG6, pHD7, pNN7, pNG8, pNN9, pNI10, pNG10, pLR-NG, p_FusB7, p_FusB8, pNN8 and pFUS_B5.
  • Check the transformations of pHnCBS1D. We have transformants.
  • The day was cut short because of Diana’s farewell party,.
June 5th
  • Run gel with the Gibson PCRs of C.LG, C.GL, C.GS and Homologous recombination. Most of them were fine. The ones that failed were some of the Homologous Recombination and the backbone of C.GS.
  • Cut bands and do gel purification.
  • Gibson assembly of C.LG, C.GL and C.GS.
  • Colony PCR of Golden Gate Assembly. We are still not getting the expected results.
  • Growth curve of E. coli. To start working on the inductions.
  • Liquid cultures of pFUS B5 and the modules, arrays and last-repeat for the Nicotiana’s TALEN.
June 6th
  • E. coli transformation with Gibson assembly of C.LG, C.GL and C.GS.
  • Miniprep of single colonies from co-transformation (pHnCBS1D and C.SG) and TALEN’s plasmids.
  • NanoDROP to check DNA concentration.
  • Gibson PCR with the parts that didn’t amplify correctly for the Homologous Recombination Assembly.
  • Run a gel with the PCR products of the Homologous Recombination.
  • Enzyme digestion protocol for co-transfomants pHnCBS1D + C.SG.
  • Run gel of digestion of co-transfomants pHnCBS1D + C.SG. Got good results :).
  • Watch leaves from the 15 Nicotiana plants in the microscope with UV.
June 7th
  • Stock of bacteria in glycerol from liquid cultures of single colonies of pHnCBS1D + C.SG.
  • Check Gibson Assembly transformants of C.LG, C.GL and C.GS (old backbone). Didn’t work as always.
  • Tried to find out what the hell is going on with Day 1 of the Golden Gate Assembli for arabidopsis and Nicotiana.
June 10th
  • Lab organization. Found out all the minipreps with really low concentrations and did them again.
June 11th
  • Tried to do the induction of pHnCBS1D + C.SG but we didn’t reach the required OD at a sensible time.
June 12th
  • Induction of co-transformants of pHnCBS1D + C.SG from liquid cultures.
  • Find out the mistake in the backbone of C.GS.
  • Do new Gibson PCR of the backbone and the promoter of C.GS.
  • Gibson Assembly of C.LG, C.GL , C. GS and Homologous recombination vector (parts that were done from C.GS template).
  • Transformation of competent cells with the Gibson Assembly of C.LG, C.GL and Homologous recombination vector.
June 13th
  • Run gel with the Gibson PCR (backbone and promoter of C.GS ).
  • Gel purification and nanodrop of Gibson PCR (backbone and promoter of C.GS ).
  • Liquid cultures of the Gibson Assembly of C.LG. The only one with transformants.
  • Plate the plasmids from one of the sequences for Nicotiana’s TALEN.
  • Watch Nicotiana leaves (plants 1, 2, 8 and 15) in the microscope and WTs as negative control. Also Arabidopsis roots.
June 14th
  • Gibson assembly of C. GS.
  • Transformation of competent cell with C.GS.
  • Miniprep of the liquid cultures of Gibson Assembly of C.LG.
  • Digestion of C.LG.
  • Liquid cultures of the TALEN’s colonies.
  • Do Gibson PCR of C.GL (backbone+promotor) + C.GS (backbone+promotor) + backbone of HR (with a new template).
  • Run gel of Gibson PCR. Didn’t work.
  • Run gel of the C.LG digestion. Tried to run 3 gels (at the end we figured out that the comb was not working correctly ¬¬).
June 15th
  • Run gel with the digestion of C.LG. We got the expected sizes.
  • Glycerol stock of liquid cultures of TALENs.
  • Check out the plates with the transformation of C.GS. We have Colonies!!.
  • Check and tend the Tobacco plants (Suspiciously, they appear again in the Lab (we think that the termites are becoming real!).
June 17th
  • E. coli co-transformation with pHnCBS1D and C.LG.
  • LIquid cultures of C.GS.
  • LIquid cultures of TALENS pNG1 / pHD6 / pHD1 / pNG6 / pNG8 from the plates.
  • Gibson PCR for Homologous Recombination. Backbone didn’t work.
June 18th
  • Check out the plates with co-transformation with pHnCBS1D and C.LG. We have lots of colonies.
  • Make liquid cultures from co-transformation with pHnCBS1D and C.LG.
  • New Gibson PCR of backbone of RH (New primers strategy).
  • Cut gel of backbone of RH.
  • Gel purification of VirD3, Cm, VirD1 and backbone.
  • Colony PCR of colonies from C. GS → everything wrong but most likely because of the colony PCR and not because the plasmid was wrong.
  • Nicotiana DNA genomic extraction.
  • Glycerol stock of liquid cultures of TALENs.
  • MIniprep of Talens.
  • Miniprep of C.GS.
June 19th
  • Transformation of competent cells with Gibson Assembly of Homologous Recombination.
  • Miniprep of C. LG + pHnCBS1D (from the same liquid cultures as the ones used in the induction).
  • Digestion of C.GS. Good!.
  • Run gel with the digestion of C.LG.
  • Streak out one colony of agrobacterium (strain GV3101) in a plate with Gentamycin +Rifampicin antibiotics.
  • Make TOP10 E. coli competent cells.
June 20th
  • Check out plates with Homologous Recombination vector. Nice and pretty colonies.
  • Make liquid cultures and Colony PCR of Homologous Recombination vector. Worked!.
  • Glycerol stock of C.LG + pHnCBS1D.
  • Co transformation of C. GS + pHnBCBS1D.
June 21th
  • Induction of C. LG + pHNCBS1D (arabinose and IPTG). Got amazing results again :).
  • Glycerol stock and miniprep of yesterday’s Colony PCR of RH.
  • Resuspend primers of RFP strategy.
  • Check Nicotiana’s roots on the microscope for GFP activity. They have!.
  • Genome extraction for Nicotiana WT, Nicotiana and Arabidopsis.
  • PCR for mGFP5 in Nicotiana.
June 22th
  • Gibson PCR for C. GL strategy using C. GS as template (two parts needed amplification: Rubisco L and the backbone+promoter+GFP).
  • Run gel of Gibson PCR for C.GL stategy. The bands weren’t purified: there was something on the negative, some weird contamination, so the results weren’t reliable.
  • Run gels of Gibson PCR of carbo with mRFP (amplification didn’t work).
  • Digestion of Talens (newest minipreps).
June 24th
  • Liquid cultures of C.GS + pHnCBS1D → They grew beautifully.
  • Run gel with digestion of TALEN’s plasmids.
  • Liquid cultures of Agrobacterium from plates made on wednesday 19th.
June 25th
  • TALENs Day 2 (Colony PCR for white colonies).
  • Colony PCR from liquid cultures of C.GS + pHnCBS1D.
  • Glycerol stock of C.GS + pHnCBS1D.
  • Miniprep of C.GS + pHnCBS1D.
  • Liquid cultures of C.GS + pHnCBS1D for induction tomorrow morning.
  • Repeat Gibson PCR for C.GL.
June 26th
  • Run gel with C.GL and C.RFP. Didn’t work.
  • NanoDROP of C.GS + pHnCBS1D. Nice concentrations =).
  • Digestion of C.GS + pHnCBS1D.
  • Digestion of Homologous recombination vector.
  • At the end we decided to do PCR for the C.LG + pHnCBS1D instead of digestion to corroborate both plasmids.
  • Replate Agrobacterium colonies to see if Cm kills it or not.
  • Create DNA linear fragment for homologous recombination in Agro.
  • New C.RFP Primer design.
  • Woow, we really did some stuff this day!.
June 27th
  • Run a sample of PCR (RH) product on a gel with molecular weight markers to confirm size.
  • Prepare Agrobacterium for Electroporation (make liquid cultures).
  • Habemus TALENs! from the re-streaking. They look wonderful and amazing!.
  • Liquid cultures of white colonies from TALEN’s plates.
  • Send G.GS and C.LG for sequencing.
  • Nicotiana GFP transplant.
  • Touchdown PCR of backbone for C.GL → using C.GS as template.
  • THERE IS A CONTAMINATION → SOMETHING IS PRODUCING A BAND OF 1KB.
June 28th
  • Genomic DNA extraction of Nicotiana.
  • PCR for nicotianas controls (primers: mgfp5).
  • Colony PCR of TALEN’s.
  • Run gel of the touchdown PCR (backbone for the C.GL). The PCR didn’t work.

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