Team:Macquarie Australia/WetLab/Notebook

Wednesday 5/2/14 Western Blot: Western blot for ChlI1 and plasto were carried out. Results: The plasto lanes did not show any expression, however ChlI showed good expression in lanes 2,4 and 5. Thursday 6/2/14: Gibson Assembly of ChlH: G1: 3uL P1-G2 exosap: 0.5uL G3-P2 exosap: 4.8uL G4-G5-G6 exosap: 1.0uL Cam vector: 29uL with Gibson mix: 12.3uL or AMP vector 1.4uL with Gibson mix: 10.8 uL Plated out PCR of Gibson Assembly product for ChlH: Standed PCR x2 using BBF/ BBR/ BBVF2, BBVR. Results: February week 2 Wednesday 12/2/14 Nanodrop of ChlH fragments ChlH Fragments: P1-G2: A= 19.3 ng/ml B= 23.4 ng/m C= 18.2 ng/m G3-P2: A = 27.8 A= 141ng/ml B= 9.9 ng/ml C= 47.5 ng/ml G4-G5-G6: A= 141ng/ml B= 24.6 ng/ml C= 62.5 ng/ml ChlD Fragments: D1, D2, D3 Thursday 13/2/14 Gel Electrophoresis for ChlH and ChlD fragments: Top Gel Lane order: 1- Ladder, 2- G1, 3-5 - P1-G2, 6-8- G3-P2, 9-11- G4-G5-G6, 12-14- ChlD Bottom Gel Lane Order: 1+ 2- Ladder, 3-5- P1-G2, 6-8- G3-P2, 9-11 G4-G5-G6 Results: ChlH fragments appear not have been digested. P1-G2 and G4-G5-G6 didn’t have plasmids on the gel so they did not digest. ChlD has digested with MLU and partially APAI. ChlD plasmids from lanes 12 and 13 were added together for further re-digestion. New Digestion using E+P from previous gel electrophoresis for ChlH: Lane Order: 1- G1, 2- G3-P2 A, 3- G3-P2 B, 4- G3-P2 C, 5- G4-G5-G6 A, 6- G4-G5-G6 C, 7- Plasto Control New Digest for ChlD with APAI: The ChlD being redigested is a combination of lanes 12 and 13 from the previous electrophoresis gel. ChlD Ligation : ChlD was ligased and transformed into E.coli. 2 colonies grew on the 300uL plate and 1 colony on the 30uL plate. Friday 14/2/14 Plasmid nanodrop Lac Gun A 33.8 ng/µl B 17.8 ng/µl Lac ChlM A 33.5 ng/µl B 26.8 ng/µl Lac ChlP A 32.2 ng/µl B 14.4 ng/µl Lac ChlI2 A 18.5 ng/µl B 15.2 ng/µl Lac POR A 35.1 ng/µl B 24.5 ng/µl Lac ChlG A 8.3 ng/µl B 13.2 ng/µl Lac YCF54 A 29.3 ng/µl B 33.9 ng/µl Lac CTH1 A 59.1 ng/µl B 51.6 ng/µl ChlH re-digest: Digests checking biobricks: With/without lac Biobrick Fragment Expected weight Measured Lac Gun A 930 ~930 B ~930 Lac ChlM A 873 ~1050 B ~1050 Lac ChlP A 1299 ~1500 B ~1500 Lac ChlI2 A 1212 ~1400 B ~1250 Lac POR A 1067 ~1250 B ~1250 Lac ChlG A 1050 ~1250 B ~1250 Lac YCF54 A 471 ~650 B ~650 Lac CTH1 A 1152 ~1350 B ~1350 DVR1 A ~1106 B ~1106 C ~1106 D ~1106 Results: Majority look as though they match the expected band length. All digests excluding DVR1 include lac. AUGUST (SEMESTER 2) WEEK 1 Thursday 07/08/14 Off and running with the whole team of 12 Biomolecular Major students. We sat through a full day of learning about iGEM; we had a discussion of project goals and aims; as well as a refresher course on how the Chlorophyll pathway works. Roles were assumed by our wiki-chiefs and those interested in gaining sponsorship and promotional roles were also filled. The wet lab group started to discuss the plan for the first wet lab next week as well as looking over the protocols. WET LAB WEEK 2 Thursday 14/08/14 Project Name: After brainstorming many names, we decided on "The Green Machine" as our title and the slogan "Follow the biobrick road" as our theme to carry throughout our wiki page. Stocks: Made many stocks, plates and buffers as per methods. Nanodrop: leant how to use the nanodrop to quantitate all parts Gene Info: We did another stock-take of parts & checked that we had enough to perform ligations to assemble or planned three Operons. We discussed the strategy for how we were going to make each of the three Operons. The parts we require to assemble our pathway are as follows: ChlD - 2240bp ChlI1 - 1202bp ChlI2 - 1298bp GUN4 - 782bp ChlH - 4207bp CTH1 - 1382bp YCF54 - 556bp Plasto - 410bp ChlM - 959bp POR - 1154bp DVR1 - 1193bp ChlP - 1385bp ChlG - 11366bp ChlD BioBrick Correction As before, previous BioBrick (BB) from 2013 had a 50bp deletion/error within the ChlD (900bp) from using a single restriction digest. Our Aim is to excise the entire ChlD gene using Apa1 and Mlu1 restriction enzymes and to insert a complete ChlD gene into a BB. We attempted this experiment again but did restriction enzyme digestions with the two enzymes separately to improve efficiency of cutting. To prevent re-joining after digestion of our cut vector, we treated our samples with Alkaline Phosphatase (Fast A.P.). The DNA was then run on a 1% Agarose gel. 5 bands were identified and using a 1Kb ladder a complete ChlD (900bp) band was found and excised for ligation. WET LAB WEEK 3 Thursday 21/08/14 More plasmid prep was done, the following 6 genes were inserted into an Ampicillin backbone. Cells were grown to extract more plasmid for stocks. Chl1 Chl2 YCF54 ChlP DVR1 POR Composite Part Assembly: Trouble-shooting with the BioBrick assembly protocol, we found that if we ligated in a particular way then the plasmid linearises itself and then cannot be cut for the making of composite parts. We then started working on forming test composite parts. Our stocktake was also completed. Composite parts were assembled of: lac + GUN4 + ChlI1 ; lac + GUN4 + ChlI2 ; lac + ChlI1 + GUN4. Digests were done with EcoRI & SpeI on first gene in part (with lac), and separately with XbaI and PstI for the second gene. These were then ligated into a KAN backbone which was digested with EcoRI & PstI. Digests and ligation steps ran at 37C for 1h and 80C for 20 minutes. DH5-a electrocompetent E.coli were transformed via electroporation and plated out onto KAN LB-agar. Transformations: Electroporation does not appear to be working well. We changed to heat shock to transform our cells. There may be a problem with our electro-competent cells. Made more electro-competent cells to test. Also made chemical competent cells for heat-shock transformation DRY LAB WEEK3 Thursday 21/08/14 Decided on Outreach ideas. FINALLY. Online reality contest “So You Think You Can Synthesise”. Had discussions of framework for competition, making a trailer. Met up with MQ Media Team later during the week Started to put together the sponsorship package WET LAB WEEK 4 Thursday 28/08/14 Digests Digests of GUN4+ChlI2 & ChlD to check results from last week Competent cells Electroporation does not seem to work and create viable competent cells ergo we shall stick to the heat shock methodology for further preps. More cells were made and used for plasmid preps, BB’s and composite parts. Composite Parts 4:1 insert – vector ratio for Fast AP ligation steps to produce: AMP backbone CTH1 + YCF54 CTH1 + Plasto ChlP + ChlG CAM backbone ChlD KAN backbone GUN4 + ChlI2 GUN4 + ChlI1 ChlI1 + GUN4 WET LAB WEEK 5 Thursday 04/09/14 A busy week! Composite parts that had growth were digested with X & P and run on 1% Agarose gel to check insert size. PCR was also performed with BioBrick Forward and BioBrick Reverse primers to see if we could confirm correct assembly of composite parts. further composite parts were assembled on the AMP backbone ChlM + YCF54 POR + DVR1 POR + ChlP Composite part RE digest images from plates that had growth Friday 05/09/14 Liquid cultures of composite part transformants Plasmid preps RE digest of each part – into CAM BB Competent cell prep: both chemical and electro-competent cells were made New composite parts made: /ChlM+YCF54/ /POR+DVR1/ /POR+ChlP/ WET LAB WEEK 6 Wednesday 10/09/14 Ran PCR products from last week ChlD CTH1 + YCF54 CTH1 + Plasto ChlI2 + GUN4 GUN4 + ChlI2 Plasmid preps done Thursday 11/10/14 Sequencing Plasmids were positive, sent to Macrogen for sequencing: CTH1 + YCF54 ChlD Gun4 + ChlI1 CTH1 + Plasto Composite Checks These composites were cut as a single digest and a double digest, and run on an agarose gel. Sizes were compared, POR + ChlP POR + DVR1 ChlM + YCF54 CTH1 + Plasto Composite part screenings Composite Parts New composite parts were made, and built upon, transformed and plated out: /CTH1+YCF54/ + Plasto /CTH1+YCF54/ + ChlM ChlI2 + ChlI1 ChlI2 + GUN4 Open Day Saturday 13/09/14 Set up chromatography reactions in preparation for open day on Saturday. Fluorescent plates drawn, grown, ready to go for Saturday. Plasmid prep done for previous composite parts. CHlH has finally worked WET LAB WEEK 7 Monday 15/09/14 Nanodrops of plasmid preps. single and double digests of every second plasmid (a, c, e, f) run gel. Send for sequencing if successful. Wednesday 17/09/14 gels re-labelled transformations CTH1 + YCFS4 + Plasto <- ChlM GUN4 + ChlD + CHlI2 Thursday 18/09/14 New Composites POR + ChlP + ChlG POR + DVR1 + ChlG POR + DVR1 + ChlP ChlD All transformed and plated out. ChlD Fix Apa1 & Mlu1 digests with the backbone being treated with Fast AP in Mlu1 digest reaction. Ligations was as usual. Then transformed and plated out onto CAM plates. Gel digests were run to resolve the 50bp difference (850-900). The resolution was seen. ^ Colony screen apaI mluI ChlD 850 and 900 + ChlH ^ pET ApaI MluI Digest Gel ChlH PCR reaction was also run WET LAB – MIDSEM BREAK W1 Monday 22/09/14 Liquid Cultures from Thursday plates. POR + ChlP + ChlG POR + DVR1 + ChlG POR + DVR1 + ChlP ChlD Restriction Enzyme Digest of CTH1 + YCFS4 + Plasto + ChlM + ChlD composite part. Gel run of ^ composite part and ChlD to compare with pET to confirm to presence of the 50bp. Tuesday 23/09/14 EcoRI + X/P digests for plasmids from Mon. ChlD A + M individual digests, compared against pET with same digest. Re-screen CTH1 + YCFS4 + Plasto + ChlM colonies and grow in liquid culture Wednesday 24/09/14 Recheck: ChlH in KAN and CAM both were not okay. CTH...ChlM: not okay therefore re screen plates from liquid cultures. Composite part-> POR + DVR1 + ChlP + ChlG Transform: ChlI2 + GUN4 + ChlD} new composite and plate out. Rescreen plated samples of CTHI + Plasto & ChlM + YCFS4 all in AMP and put into liquid culture. Thursday 25/09/14 Liquid culture of ChlI2 + GUN4 + ChlD Plasmid prep done in afternoon POR ...ChlG transformed and plated out CTHI ...ChlM gel resolved and excited for friday purification. ChlI2 + GUN4, CTH1 + YCF54 and POR + DVR1 into CAM backbones. prepare ChlI2 + GUN4, CTH1 + YCF54, ChlD for sequencing Plasmid prep of CTHI + Plasto, ChlM + YC5S4. Friday 26/09/14 CTHI ..ChlM gel band purified Liquid cultures of new composite parts (ChlI2 + GUN4 + ChlD POR..ChlG) Plasmid prep of CTHI + plasto (A-D) and ChlM + YCF54 (A-D) Restriction enzyme screen plasmid prep from thursday. Transformation of GEl extracted plasmids (linear [total of 10 for transformation] + circular + ligation) *1ul ligase and 4.5 ul ligase buffer. 37oC for 1 hour and 80oC for 20mins. → 5ul linear plasmid → 50ul competent cells (chemical) → 10ul circular plasmid → 50ul competent cells Gel Run for ChIl2 + GUN4 + ChlD, ChlH1 + Plasto & ChlM + YCF54 cuts. LANE ORDER: 1.1….13.2- ChIl2 + GUN4 + ChlD - ChlH1 + Plasto - ChlM + YCF54 *note: The wells for the last 3 lanes did not accept much of the sample. The sample would float to the surface when being inserted. The transformants were plated out, the digest resolution wasn’t clear and needs to be re run on monday. liquid cultures of Sunday transformants 28/9 (CTH1...ChlM gel purified CTH1 + YCF54 (CAM), ChlI2 + GUN4 (CAM). WET LAB – MIDSEM BREAK W2 Monday 29/09/14 plasmid preps of: ChlM CAM CTH1..ChlM gel purified CTH1..ChlM re-screen POR..ChlG POR + DVR AMP CTH1 + YCF CAM ChlI2 + GUN4 CAM Re Digest and Gel which all results were good: ChlI2 + GUN4 + chlD CTH1 + plasto AMP ChlM + YCF54 AMP Gel: ChlH linearized and gel purified. Digest + gel: CTH1..ChlM and POR...ChlG CTH..ChlM plasmid prep kept to H, I, J POR ..ChlG plasmid prep kept to 1:1, 1:3, 1:6 ChlH upper and lower bands excised & gel purified Tuesday 30/09/14 Re-transformed and plated out: CTH..ChlM x 3 POR..ChlG x 3 ChlH x 2 CTH..+ POR.. mixed x 2 Gel run on CAM transformants and re-screen of: POR + DVR, ChlI2 + GUN4, CTH + YCF Composite part created, transformed and plated out: ChlI2 + GUN4 + ChlD + ChlI1 ChlM cut into CAM Backbone, transformed and plated out. Tested for registry PCR reactions run on final constructs and checked for F &R from ends: CTH...ChlM POR...chlG Wednesday (1/10/14) PCR: 1ul BB 5ul buffer (x10) 1ul F primer 1ul R primer 1ul dNTP 0.25 ul Taq 0.75 ul H2O (to Evel 50ul) Master Mix: 50ul buffer 10ul dNTP 2.5ul Taq 407.5ul H2O ------ X ------ C1 + C2 → CTH1 + ChlM (H) (CTF + BBR) (CMR + BBR) C3 + C4 → CTH1 + ChlM (I) (CTF + BBR) (CMR + BBR) C5 + C6 → CTH1 + ChlM (J) (CTF + BBR) (CMR + BBR) PI → POR A P2→ PORB P3 → PORC -----X----- Setup for functional assays and plasmid preps of ChlM & Chli1 + Chli2 Thursday 2/10/14 Cyclase assay (100ul) 10uM MPE - 8ul 1mM NADP - 10ul 10mM G-P-P - 10ul Assay buff 1 x (50mM tricine, 2mM MgCl2, 1mM DTT 10% glycerol, pH 8.0) - 50ul 2x 0.5ul of G-6-P-dehydrogenase total volume = 78ul allowing 22ul for other additions. #1 Blank (water) #2 22ul CTH1 part 1 #3 11ul CTH1 part 2 #4 11ul CTH Bradford functional assays were done on induced cell pellets 10% Glycerol + 5mM Tricine NaOH ph8.0 + 2mM MgCl2 + 1mM DTT Results: 5µl 2.5 dilution POR - ChlG 4 ~ 1.25 1 3 ~ 1.75 1.5 2 ~ 2 1.5 1 ~ 2 1.5 CTH - ChlM 1 ~ 1 1 2 ~ 1 1 Friday 3/10/14 Protein weight estimates: ChlM - 30440 Da CTH1 - 43873.3 Da YCFS4 - 17073.7 Da Plasto - 10339 Da Chli 1 - 39952 Da GUN4 - 2450.6 Da ChlD - 76420.1 Da POR - 41871 Da DVR1 - 37034 Da ChlP - 47011 Da ChlG - 36880 Da Protein gels run of two complete composites. ^CTH1-ChlM composite ^POR-ChlG composite The CTH1-ChlM composite showed good separation of products. The POR-ChlG composite had separation but only three parts were able to be easily identified. As Annotated the selected bands were cut-out for in-gel digestion & analysis by MALDI- TOF/TOF. WET LAB WEEK 8 Wednesday 8/10/14 Plasmid preps were done on the Chli1 - ChlD composite parts Nanodrops: Sample nucleic acid conc (ng/µl) 260/280 Chli1 + ChliD 179.6 1.92 Chli1 + ChliD 296.3 1.87 Chli1 + ChliD 261.7 1.93 Chli1 + ChliD 648 1.90 Chli1 + ChliD 118.7 2.07 Chli1 + ChliD 179.1 1.79 Chli1 + ChliD 553.7 1.93 Chli1 + ChliD 321.6 1.92 All 8 were then digested and run on a gel, 5-10mL liquid cultures were also prepared and left to incubate overnight. 5-10mL liquid cultures were made of the following parts in an AMP backbone with a lac promoter. Chli2, YCFS4, ChlP, POR, GUN4, ChlM, ChlG, CTH1, CTH1-ChlM, POR-ChlG for large scale growth (50mL) for functional assays. Thursday (9/10/14) New composite Chli1 + ChlD + GUN4 transformed and plated out. Intermediates and final parts prepped and sent for sequencing to confirm that the inserts are what they’re supposed to be. Gylcerol stocks were made of the current intermediates and final composites. Final parts in an AMP backbone were induced (OD600 = 0.4-5) for functional assays. WET LAB WEEK 9 Monday 13/10/14 Plasmid prep of Chli1+ChlD+GUN4, All cultures were screened and prepped for sequencing. Nanodrops: Sample nucleic acid conc (ng/µl) 260/280 Chli1+ChlD+GUN4 539.2 1.86 Chli1+ChlD+GUN4 315.4 1.91 Chli1+ChlD+GUN4 493.7 1.90 Chli1+ChlD+GUN4 164.0 1.91 Chli1+ChlD+GUN4 217.5 1.90 Chli1+ChlD+GUN4 447.0 1.90 Chli1+ChlD+GUN4 499.2 1.90 All composites re-run on gels for results page, SDS-PAGE gels and MS/MS prep. POR-ChlG and Chli1-GUN4 parts were re-transformed for lysate harvesting. ^ Chli - ChlM Final gels ^ POR-ChlG Final gel Tuesday 14/10/14 POR assay’s were run with GUN4 activing as a negative control. We expected to see a major peak at ~630nm and a secondary peak at ~670nm, but on both runs (a 20minute and 1.5hours) the second peak was still not visible. Cyclase assays were also run mirroring experimentation from 2/10/14 on CTH1 and POR, these were run overnight with an expected peak at ~590nm showing presence of the Mg - protoporphorin intermediate. The Mg is apparent but no intermediates have been generated. Wednesday 15/10/14 Liquid cultures were made from plate cultures from the previous day (POR-ChlG, CTH1, Chli1-ChlD-GUN4, POR). The liquid cultures from the POR-ChlG & Chli1+ChlD+GUN4 composites were then induced for growth as 50mL cultures for French Pressing. The resulting proteins were then run on SDS-PAGE gels and used for functional assays; gels were destained and bands cut for in-gel digestion and MALDI-TOF/TOF. Thursday 16/10/14 ^ CTH1 - ChlM Final gel ^ POR-ChlG Final Gel Friday (17/10/14)

Thursday 16/10/14

Assaying CTH1, YCF54 and Plastocyanin

Upon building the second operon including CTH1, YCF54 and Plasto, a functional assay testing the successful or unsuccessful expression of the yielding protochlorophyllide was made. The assay mix included;

Insert table code Reagents Volume 5µM magnesium-protoporphyrin IX monomethyl ester 8µl 1 µM NADPH 10µl 10 µM glucose 6 phosphate 10µl 1 µg/ml glucose 6 dehydrogenase 0.5µl 50 mM tricine 5 µl 2mM MgCl 5 µl 1mM DTT 5 µl

Extract from chlamydamonas was added to; 1. Supernatant + membrane (1:1) 2. Supernatant only 3. Membrane 9 CTH1 supernatant samples were assayed, followed by the addition of 5 µl of water and centrifuged and was run on HPLC for 1.5 hrs. No significant protochlorphyllide levels were detected using this assay, this technique however may be used in future assays,

Friday 18/10/14

Assaying the second operon CTH1, YCF54, Plasto including ChlM

Including the final component necessary for the production of protochlorophyllide in the second operon was the addition of ChlM. The assay mix was added to the pellet and was as follows;

Insert table code Reagents Volume Resuspension buffer Made to 44 µl SAM 1mM 1 µl MgP 20 µM 2 µl Insert image code

Figure x shows the MP standard elutes at 9.8 minutes, the precursor to the catalysis to MPE

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Figure x shows the MPE standard shows a peak at 10.4 minutes

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Figure x depicts the SAM control shows a significant peak at 9.8 minutes showing it does not result in the MPE peak at 10.4 minutes.

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Therefore, figure x shows the assay presents a peak at 9.7 minutes. Presence of MP and the absence of MPE indicates the lack of conversion of MP to MPE. This may be due to a low level of expression or low ChlM activity.

� LAB BOOK - PROTOCOLS PLASMID PREP Centrifuge @13200 rpm for 10min 1.8mL of overnight cultures in 2mL eppendorfs Discard supernatant and add 1.9mL of culture and centrifuge again @13200 rpm for 10min Resuspend pelleted cells in P1(250µl) add 250µL of P2 invert 4-6 times (turns homogenous blue) Add 350µL of N3 & mix by inverting (turns colourless) Centrifuge @ 10min 13000 rpm to create a pellet Transfer supernatant in QIA Prep Spin Column by pipetting Centrifuge 30-60sec - discard flow through Wash Qia Prep Spin Column with 0.5mL of PB Centrifuge for 30-60 seconds - discard flow through Wash spin column by adding 0.75 mL PE buffer Centrifuge for 30-60 seconds -> discard flow through and centrifuge for a further 1min Place QIAPrep Column in clean eppendorf Elute DNA add 50µl of water, stand for 1min, centrifuge for 1min QIA prep -> Spin miniprep buffer BIOBRICK ASSEMBLY Digest Prepare the following Upstream Plasmid Prep (U) Downstream Plasmid Prep (D) Destination backbone (P) NE Buffer 2 BSA 3 PCR tubes - labelled (U/D/P) Add 250ng of each part to its respective tube adjust total volume to 21.25µl with water Add 2.5µL of NE Buffer 2 to each tube Add 0.25µL of BSA to each tube RE digest - total volume ~ 50µl (U) 0.5µL EcoR1 - HF + 0.5µL Spe1 (D) 1µL Xba1 + 1µL Pst1 (P) 1µL EcoR1-HF + 1µL Pst1 Mix & Spin Down Incubate @ 37oC ~ 15min then @ 80oC for 20min [OPTIONAL] Run a gel with 20µL of each [OPTIONAL] Store @ -20oC Ligation Prepare the following 10x T4 DNA ligase Buffer T4 DNA Ligase PCR tube (L) Add 11µL of water to the L tube Add 2µL from each digest tube (U/D/P) Add 2µL of 10x Buffer Add 1µL of T4 Ligase total volume = 20µL Incubate @ room temperature for 10min Incubate @ 80oC for 20min Store @ -20oC or transform. FAST AP LIGATION HEAT-SHOCK TRANSFORMATION Add 2µL ligation reaction to chemically competent cells and mix Incubate on ice for 30min Heat shock @ 42oC for 30sec Store on ice for 2min Add 950µL of SOC medium Incubate @ 37oC for 1hr with shaking Plate out 5-200µL onto LB plates w/ antibiotic required & IPTG if necessary Incubate overnight @ 37oC ELECTROPORATION TRANSFORMATION Add 1µL ligation reaction to a fresh PCR tube Add 50µL electroporated cells pipette mix into an electric cuvette and pulse Add 1µL warmed LB broth to cuvette and mix gently Quickly transfer contents to a fresh tube Incubate @ 37oC for 1hr.