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.

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.

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 °C for 1 hour

- 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)

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

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.

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.

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

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.

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.

Monday

- Transformed Agro for Canary 1 experiments and spreadplated onto LB plates containing Rif, Gen and Amp antibiotics and stored at 28 °C for 2 days. - Infiltration of Agro constructs 2,3,4,10,11 into N. Benthamiana and plants stored in Plant Growth room at UEA O/N. - Cells from Flipper cultures harvested and frozen at -20 °C O/N.

Tuesday

- Mini prep of all 4 Mo flipper modules (PRO, CDS, TER CGCT) - Nanodrop quantification- concentration of DNA not great enough for sequencing - O/N cultures of Mo flippers - Discussions with head of school regarding authorised absence for iGEM

Wednesday

- O/N cultures of agro level 1s and 2s - Mini prep of O/N cultures of mo flippers, quantification of DNA - acceptable amount to be sequenced

Thursday

- Mo flipper DNA prepared and sent for sequencing - Glycerol stocks of agro level 1 and 2 constructs - Meeting with Michael Pfeil regarding ethics approval for surveys - Set up 100 mL cultures for all four Flippers - Checked infiltrations under UV but no fluorescence observed

Friday

- Midiprep of overnight Mo flipper cultures from wk10 d4. - Quantification of DNA from Mo flipper midiprep.