We have documented the day to day progress of our iGEM project in our online Lab Notebook, which includes our wet lab experiments, policy and practices planning and outreach events.

Week One >

Monday

- Contacted by email UEA based organisations UEA:TV, Concrete and Livewire regarding possible exposure for our project. - Emailed Sam Fountain (UEA) regarding possible sources of funding and who to approach from the University of East Anglia.

Tuesday

- Started tweeting from the UEA iGEM Twitter account. - Created the UEA iGEM 2014 Facebook page. - Emailed MustardTV regarding exposure for our project (local to Norwich).

Wednesday

- Created a poster design for the Oxford SynBio meet up held at Oxford University, - Poster printed

Thursday

- Attended Synthetic Biology 'Meet up' hosted by Oxford for iGEM teams across the country.

Friday

- Submitted 'About our lab' form for iGEM before deadline.

Week Two >

Monday

- Completed The Sainsbury Laboratory (TSL) safety induction. - Collected synthesised samples from DNA 2.0 and transformed DNA into E.coli using electrocompetent cells and electroporation. Added 500 μl of Soc broth and incubated for 40 minutes at 37 °C. Spreadplated onto agar plates containing Kanamycin and incubated O/N at 37 °C.

Tuesday

- Selected a single colony from each of the eleven plates and the colonies were grown in TY broth with Kanamycin O/N - Transformation of pSB1C3 plasmid containing RFP into E.coli using electroporation. Incubated with 50 μl of Soc broth at 37 °C for 1 hour. - Spreadplated E.coli cells which have been transformed with the pSB1C3 + RFP plasmid. - Completed calculations for Dig-Lig experiments (Size and conc. of DNA parts calculated,size of acceptor calculated, order of constructs for each reaction determined, dilution and DNA conc. for each reaction determined) Ratio of 2:1, constructs:acceptor (plasmid).

Wednesday

- Midi prep following the Qiagen protocol of the 9 constructs previously synthesised by DNA 2.0 and transformed on Day 2 and DNA conc. of each constrcut determined using the nanodrop. Dig-Lig calculation spread sheet completed using these concentrations. - Set up the first 11 Dig-Lig experiments, placed into PCR block on the slow protocol. - Carbenicillin plates made and spreadplated with 40 μl of XGAL and 100 μl of IPTG. Plates left to dry upside down in 37°C incubator. - Transformation of GG DNA into electrocompetent E.coli cells, PROTOCOL: • Cells removed from -80°C freezer and allowed to thaw. • A 5 µl sample of DNA was added to a 50 µl aliquot of cells. • Transfered each 55 µl sample into a cuvette. • Electroporation pulse on setting ECR1. • A 500 µl of SOC broth was added to the cuvette. • A 555 µl cuvette sample was transferred to an eppendorf tube and incubated for 40 min at 37°C. • A 100 µl sample was spreadplated onto agar plates containing carbenicillin and incubated O/N at 37°C. - A 20 µl PCR reaction was set up using a GG level 1 acceptor as template DNA containing the primers P1: 0349(PRO+5U) and 0353 (3U+TER) and P2: 0350 (PRO+5U) and 0354 (3U+TER). - A TBE buffer 1% agarose gel was poured for diagnostic purposes to check the amplification of the PCR products. - PCR product purification • Pre-percipitation to remove TAQ polymerase and protein was completed followed by phenolchloroform extraction (x2). Aqueous layer is removed and discarded leaving DNA. • Precipitation to remove buffer was carried out by adding Na acetate (10%) and ethanol causing the DNA to come out of solution. Aspirator was used to remove buffer, salt and ethanol. • A salt wash was completed to remove the acetate and ethanol. • Sample was incubated at 65°C for 2 min to evaporate the ethanol.

Thursday

- Checked Dig-Lig tranformation plates grown O/N and poor blue white selection observed with many blue colonies. - Selected 2 white colonies from each plated and colony PCR performed. - Following colony PCR, amplified fragments ran on gel to determine size and therefore whether they contained the construct. - Decided to repeat the 11 Dig-Lig experiments - Ran digested PCR product from 25.06.2014 on a diagnostic 1% agarose gel in TBE buffer and determined that LacZ had internal restriction enzyme cut sites and product was split into two bands.

Friday

- Found poor blue/white selection on dig-ligs from 26.06.14. Suspected to be due to either the enzyme used or PCR block. - Set up a new set of dig-ligs to test the source of this problem.

Week Three >

Monday

- Set up 10 Dig-Lig reactions with new BSA1 enzyme provided by TSL. - DNA was transformed into E.coli using electroporation method previously discussed. - Transformed bacteria was spreadplated onto canamycin plates containing XGAL and IPTG and incubated O/N at 37°C. - Mini-prep of DNA obtained from Week 2's successful Dig-Lig experiments according to Qiagen protocol. However, DNA eluted in 25 µl of eluting buffer rather than 50 µl as protocol suggests. - DNA conc. of each sample quanitifed using the Nanodrop. - DNA diluted to obtain 5 ng in each 5 µl sample. - Sense and Antisense primers to each 5 µl DNA (+H20) to achieve 20 µl sample in total and sent for sequencing to verify construct sequence.

Tuesday

- Selection of 2 white colonies from 30.06.14 Dig-Lig transformed plates for a colony PCR reaction. The same 2 colonies were grown in 5 ml of SOC broth O/N at 37°C. - Poured a 1% agarose gel using TBE buffer with a 1:20,000 dilution of ethidium bromide. - Gel run with Dig-Lig PCR products.

Wednesday

- Transformed 5 constructs into Argobacterium tumefacians • Mini prep of 5 constructs from colonies picked 1.7.14 • Electroporated to transform into agro • Incubated at 28 degrees for 1 hour - Poured plates - Spread plated transformed agro

Thursday

- Set up a 20 µl PCR reaction using primers synthesized from Sigma and a level 1 acceptor as the template DNA • Pro + 5UTR • CDS • Ter + 3UTR - Ran PCR product on a gel to check amplification - PCR product purification using phenol chloroform

Friday

- Digest of PCR samples from 3.7.14 - Gel purification and quantification of digested fragments (now ready to ligate into PSB1C3 backbone) - Digest of PSB1C3 plasmid with EcoR1 and PST1 (removes blunt ends) - Dephosphorylation of plasmid backbone - Colony PCR of colonies picked from agro plates (transformed 10.7.14)

Week Four >

Monday

- Colonies picked from agrobacterium plates from 2.7.14 - Overnight culture in 5 ml of broth containing Carbenicillin, Gentamicin, Rifampicin

Tuesday

- No wet lab work completed - Team meeting to discuss human practices events we would like to organise

Wednesday

- Safety induction in UEA labs for all team members - Human Practices meeting. Ideas included: Food security event, surveys, School events and possible speed debate.

Thursday

- Agrobacterium-mediated transient expression in leaves of N. benthamiana PROTOCOL: 1. Single colony of each constructs 2,3,4,10,11 grown overnight at 28 °C in 5 ml media containing chloramphenicol. 2. Pellet by centrifugation at 4000 rpm for 15 min. 3. Resuspend in 10 mM MES, 10 mM MgCl2 and 150 μM Acetosyringone. 4. 1:100 dilution to give a final OD of 0.1-0.5 5. Incubate at RT for 4 hours. 6. Create a small puncture using a needle on the underside of N. benthamiana leaves of plants 6-8 weeks old. Using a blunt syringe infiltrate the agro constructs into the leaf puncture. Each construct is infiltrated into 4 sections on one leaf. 2 constructs per plant. 7. Store plants in controlled environment at 19-23 °C for 3 days. -Ligation reaction to join PSB1C3 and GG compatible ends.

Friday

- Analysis of infiltrations using light microscope and UV light. - Colonies picked from ligation on 10.7.14 (GG compatible PSB1C3 for accepting Pro, CDS, Ter level 0 parts) and grown in 5 ml of LB broth, incubated in shaker O/N at 37 °C - Attended the UEA BIO Research Colloquium from 9am - 3.30pm

Week Five >

Monday

- Mini prep of Mo Flipper Modules DNA - Quantification of DNA (Nanodrop) PRO 1 - 78.5 ng/µl PRO 2 - 93.5 ng/µl PRO 3 - 73.8 ng/µl CDS 1 - 77.7 ng/µl CDS 2 - 71.1 ng/µl CDS 3 - 66.8 ng/µl TER 1 - 91.8 ng/µl TER 2 - 89.2 ng/µl TER 3 - 89.2 ng/µl

Tuesday

- Visited The CUT venue in Halesworth in preparation for the Food for Thought organised event on the 26th July.

Wednesday

- Meeting with Dr Anna Smajdor, a bio-ethicist, to discuss ethical implications of our project. - Discussed the need for ethical approval for data collection for possible ideas such as surveys.

Thursday

- Team meeting to discuss how lab work is progressing and who/when the further lab work will be carried out.

Friday

- Preparation of media required: LB, LB agar, LB + Chloramphenicol plates and LB + Kanamycin plates.

Week Six >

Monday

- 2 single colonies picked from agro plates grown 02/07/2014. - Colonies added to 5 ml of broth containing Carbenicillin, Gentamicin and Rifampicin

Tuesday

- Transformation of synthesised DNA from DNA 2.0 into E.coli. Tubes 1 to 7. 1. AtPR1 2. ArvBS3 3. NbBi1-RNA 4. NbProB-RN 5. AvrXa27 T 6. OsXa27 Pro 7. Control (No DNA) - Followed heat shock protocol: - 5 µl DNA added to 50 µl of chemically competent E.coli - Spreadplated onto agar plates containing KAN - Incubated O/N at 37°C

Wednesday

- Met with Dr Kay Yeoman, BIO outreach officer (UEA) to discuss activities for school outreach events. - Discussed activities for 'Food for Thought' event at the cut on 26/07 including visual representations of crop losses in cylinders.

Thursday

- DNA preparation of Nevada 2010 biobricks in preparation for characterisation. - O/N culture of single colonies of level 0 constructs (currently in E.coli)

Friday

- Mini-prep of O/N cultures grown on 23/07/14 of DNA synthesised by DNA 2.0. Tubes 1 to 6: 1. AtPR1 Conc: 182.2 ng/µl 260/280: 1.94 2. ArvBS3 Conc: 342.7 ng/µl 260/280: 1.93 3. NbBi1-RNA Conc: 152.2 ng/µl 260/280: 1.95 4. NbProB-RN Conc: 181.6 ng/µl 260/280: 1.90 5. AvrXa27 T Conc: 263.2 ng/µl 260/280: 1.90 6. OsXa27 Pro Conc: 286.5 ng/µl 260/280: 1.90 - DNA quantified using Nanodrop and blanked with EB buffer. - Further mini prep of O/N cultures grown on 22/07/14 and 23/07/14 from the BioBricks requested from the 2010 Nevada team's (https://2010.igem.org/Team:Nevada) project 5 mL was analysed from an inoculation of 10 mL LB + CAM. Biobricks obtained are: BBa_K414001, BBa_K414003, BBa_K414004 and BBa_K414008 - Run on a 1% agarose gel with TAE buffer. - Preparation of the 'Food for Thought event at 'The CUT' tomorrow where we will be giving presentations and demonstrations about food security - Created business cards for distribution at outreach events - Collected leaflets for distribution - Digest of PSB1C3 to drop out RFP and ran on 1% TAE agarose gel to characterise.

Week Seven>

Monday

- Meeting with Tom Shakespeare based at UEA Med to discuss the ethics of 'The Green Canary' Project.

Tuesday

- Digest of PRO, CDS and TER with Bsa1 - Set up level one dig-ligs 1, 5, 9, 15, 16, 17, 18, 19 and PCR. - Agrobacterium transformation. 2 μl DNA into 50 μl of cells followed by electroporation. Incubation in LB for 2 hours. Spreadplated onto LB containing: 1 μl/ml of Ampicillin (100 mg/mL), 1 μl/ml of Rif (25 mg/mL) and 1 μl/ml of Gentamycin (10 mg/mL). Incubated O/N at 30 °C.

Wednesday

- Made 2 L of LB Agar - Poured 8 agar plates containing Ampicillin (1 µl/ml). Spreadplated IPTG and X-GAL. Spreadplated the 8 dig-lig reactions created on the 28/07/14. Tubes 1,5,9,15,16,17,18,19. - Picked 1 colony from PRO, CDS and TER plates with Psb1c3 + RFP containing BSA1 sites to make them GG compatible to be grown in an O/N culture (37°C with shaking) in 5 ml of LB. - Picked 1 colony of the newly transformed DNA from DNA 2.0 to be grown in an O/N culture (37°C with shaking) in 5 ml of LB.

Thursday

- Picked 1 white colony from 29/07/14 transformed dig-lig plates and added to 5 ml LB + 5 μl Ampicillin and incubated at 37 °C also added to 50 μl of PCR Master Mix containing: 1 μl DNTPs 2 μl Primer 1 2 μl Primer 2 5 μl Buffer 2 μl MgCl2 0.5 μl BIOTAQ Enzyme 37 μl Water - PCR programme: Step 1 : 95 °C 5 min Step 2 : 95 °C 1 min Step 3 : 54 °C 1.5 min Step 4 : 72 °C 1 min Step 5 : 72 °C 5 min Steps 2, 3, 4 repeated x 34 times - Run on a 1 % agarose TAE gel at 100V. Each well loaded with 2 μl of loading dye and 20 μl of DNA. Two 1kb ladders loaded. - DNA mini prep of cultures from 29/07/14 (DNA 2.0 level 0's and PRO, CDS, TER) (2.5 mls) - Glycerol stocks made from 1 ml of O/N culture. - Transformations of DNA iGEM #2-19 and Vectors #1-4 intro E.coli to generate more DNA. - Made 2L of LB agar. - Met with Laura Bowater to discuss the construction of the iGEM wiki pages.

Friday

- Arranged meeting with Mark Wilkinson to discuss University regulation ethics approval for the Green Canary Project

Week Eight >

Monday

- Colony PCR of Agrobacterium plates containing constructs 2,3,4,10,11 which had been grown over the weekend at 28 °C. - Colony PCR products run on a 1% agarose TAE gel with a DNA positive control. - Picked single colonies of Level 1 constructs: 1, 5, 9, 15, 16, 17, 18, 19. Colony PCR of these level 1 constructs. - 2 Agrobacterium colonies selected from each plate to be grown in 5 ml of LB, 100 µl Ampicillin, 200 µl Gentamycin and 200 µl Rifampicin O/N at 28 °C with shaking. - 93 agar plates autoclaved and poured for Human Practices school events in September, kept in cold room at 4 °C.

Tuesday

- Added 2g of sucrose and 0.441g of MS salts to 100ml of sterile water + Acetosyringone. - Spun down agrobacterium cultures grown on 4.8.14 - Resuspended pellets in 10ml of prepared solution - Left to grow on the bench for 3 hours - Each construct infiltrated into one nicotiana benthamiana plant on two separate leaves - Mini preps of level 1 dig-ligs (1,5,9,15,16,17,18,19) - Quantification of DNA required for next level two dig-ligs.

Wednesday

- Set up level 2 dig-lig reactions (Vector, buffer, inserts, ligases) Level 2 Constructs: A= 35s_AvrBS3 + BS3_GFP B= 35s_AvrBS3 + BS3_NbHb1 C= BS3_Bax + BS3_Bi1RNAi D= 35s_AvrBS3 + BS3_Bax +BS3_BiRNAi E= 35s_Bax1 + 35s_BiRNAi F= PDF1.2_Bax1 + PDF1.2_BiRNAi G=PR1_Bax + PR1_Bi1RNAi - PCR of Level 2 Constructs (cycles: 3 cycles of
 10 minutes 37°C,
 10 minutes 16°C, 
followed by 
10 minutes 37°C, 
20 minutes 65°C
, 4°C forever) - Made 1µl/ml Streptomycin plates. Spread with 100µl IPTG and 80µl of X-gal per plate - Transformed 5µl of level 2 construct DNA from PCR into 50µl E.coli cells, electroporation of cells, spread (amount?) onto streptomycin plates, incubated overnight at 37°C

Thursday

- Charles Brearley (UEA) demonstrated the use of the epi-fluorescence microscope in the Teaching Lab. - Infiltrations from 5/8/14 did not show GFP fluoresence as expected so will need to be repeated. - Mischa discussed ethics approval for The Green Canary project with Dr Mark Wilkinson and Dr Micheal Pfiel.

Friday

- Spread the level 2 constructs A-G on spectinomycin plates with IPTG and XGAL for blue white selection as well as plating the transformation of vector pAGM8031. - Plated end linkers 2 and 3 on ampicillin and all plates incubated overnight at 37°C. - Picked colonies of Agrobacterium tumerfaciens, 2, 3, 4, 10 and 11 and grew in an overnight culture with shaking (28°C) with 400 μL LB and 1μL/mL of ampicillin. - We invited the Cambridge iGEM team up to meet us and discuss each others projects as well as future collaborations and gave them a tour of the NRP, JIC and UEA. We discussed providing Cambridge with our MoFlipper so they can submit their parts in BioBrick form as well. - Jack met with Graeme Byrne to prepare for meeting with InCrops on the 28th of August.

Week Nine >

Monday

- Picked 2 colonies of the level 2 dig-lig's transformed on friday. A, B, C, D, E, F, G and the level 2 Vector pAGM8031. Colonies grown in 1 ml of LB containing 1 μL/mL of Spectinomycin O/N. - Colony PCR of agro cultures grown for 2 days. Construct numbers 2, 3, 4, 10, 11. 4 cultures per construct were grown. Run on a 1% TAE gel with a positive control of pure DNA for each construct.

Tuesday

- Mini prep of Level 2 E. coli A-G (2 of each, e.g A1 & A2) and Level 2 vector in preparation for PCR. - Quantified all previously prepared DNA Ran 1% TAE agarose gel of Level 2 colony PCR products.

Wednesday

- Transformation of Mo Flipper modules (Pro, CDS, Ter, CGCT) by Calcium Chloride heat shock. - Spreadplated each Flipper module onto LB plates containing Chloramphenicol. - Glycerol stock made for each transformation.

Thursday

- Collected plants from TSL but unable to infiltrate as positive control was not saturated enough.

Friday

- Set up overnight cultures of all Agro. transformations (2.1, 3.1, 4.1, 10.2 and 11.1). - Set up overnight cultures of Mo Flippers, to be incubated over the weekend so they can be minipreped and sent for sequencing.

Week Ten >

Week Eleven >

Week Twelve >