Team:Cardiff Wales/Notebook/Text

09/07/2018

First day in the lab
Had safety induction and signed appropriate forms (all)
Discuss medal criteria and how the project fits in with this (all)
Discuss overall project and specific techniques (all)
Make LB broth (all)
Produce cultures to grow overnight at 37℃, A2 and 18J (all)

10/07/2018

Miniprep the colonies grown overnight (all)
Colony PCRs on Lvl 0 plates 1-9.
1= WRKY intron/ 2= SP3.5.5/ 3= SP3.5.3/ 4= BCR3.5.5/ 5= BCR3.5.3/ 6= BCR3.FULL.5/ 7= BCR3.FULL.3/ 8= ATHSPR1P/ 9= RTBVP

11/07/2018

PCR Products from yesterday run on an agarose gel.
Set up Lvl 0 reactions for the ones that didn’t have good results from the gel
Digestion, Ligation and transformation (SP3.5.3/ BCR3.FULL.3/ ATHSPR1P and 10= GUS).
Pick new colonies from plates 1,4 and 5 to redo colony PCR, then run on a gel.
Regrew the colonies from plates 2,6 and 9 overnight at 37℃.
Make competent E. coli cells. Do a transformation with 18J to test them and plate them out, grow them overnight at 37℃.
Prepare for the UK meetup- poster/presentation/worksheets for the workshops.

12/07/2018

Half the team in oxford for the UK meet-up (RC, EH, ST and EM)
Run gel from colony PCRs from yesterday (HE and AT)
Miniprep the colonies from plates 2,6 and 9 (HE and AT)
Colony PCRs using the Lvl 0 colonies (3,7,8 and 10) and then run on a gel (HE and AT)
Fresh colonies from plates 1,4 and 5 to do colony PCRs and run on a gel (HE and AT)
Perform PCR on miniprepped colonies from plates 2,6 and 9 and run on a gel (HE and AT)
Regrew colonies from plates 7, 4,5 and 3 overnight at 37℃ (HE and AT)

13/07/2018

Half the team in oxford for the UK meet-up (RC, EH, ST and EM)
Miniprepped cultures grown overnight from plates 3,4,5 and 7 (HE and AT)
PCR on the minipreps from 3,4,5 and 7 (HE and AT)
Colony PCR on colonies from plates 1,8 and 10 (HE and AT)
Run PCR products on a gel (HE and AT)
Picked 2 colonies from plate 10 that worked and grew them over the weekend (HE and AT)

16/07/2018

Miniprepped the WRKY intron cultures grown over the weekend.
Make plates containing Chloro/XGAL/IPTG and Kan/XGAL/IPTG.
iGEM white check in form for using aphids (Health and Safety).
Email Nottingham iGEM team about collaborations.
Discuss with water institute about helping other teams for collaborations.
Email Bayer for human outreach aspect.
Set up Lvl 0 reactions for GUS, ATH, and enhancer (digestion/ligation/transformation). (RC and AT)
Transform enhancer from biobrick plate 6 L14.
PCR all of the miniprepped Lvl 0 constructs to check that they are right- run on a gel.
Sent off Lvl 0 constructs for sequencing.
Grew up more colonies from previous colonies that contain correct inserts.

17/07/2018

Miniprepped colonies grown overnight.
Set up Lvl 1 reactions for successful Level 0 constructs.
Contacted Thea (European iGEM ambassador) about collaborations.
Colony PCR for Lvl 0 colonies (Enhancer, 7 and 10).
Run PCR products on a gel - there were no bands.
Discussion about logo design.
GUS, ATH and Enhancer didn't work, so re-performed Dig/Lig (RC and AT).

18/07/2018

Transformation of bacteria for Lvl 1 reactions.
Contact Fiona about risk assessment for human outreach event at Techniquest.
Meeting with Dan to discuss 3D printing for human outreach.
Transformations using the iGEM registry parts for the interlab study.
GUS,ATH, Enh cells grew but were all white. Performed colony PCR (cPCR) anyway but none worked. (RC and AT).

19/07/2018

Colony PCR for new lvl 1 plates with primers 64+69 and gel run.
Colonies with correct band sizes grown overnight.
Aphids obtained and placed onto tobacco plant.
Dig/Lig on GUS, ATH and Enh again. Transformed and grew overnight (RC and AT).
Made new plates with surface IPTG (40 microlitres of 100mM) and X-Gal (96microlitres of 25mg/ml) (RC and AT)
Transformed InterLab study constructs (RC, AT and EM)

20/07/2018

Contacted Alice about human outreach event in North Wales.
Miniprepped level 1 colonies grown overnight, then tested again with primers 39+69.
Colony PCR for level 1 plates that didn’t show bands in yesterday’s colony PCR.
More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.
ATH and GUS were pale blue so left in fridge over weekend (RC and AT).
Dig/Lig again on GUS, ATH and Enhancer. Transformed these and redid 2N and 2F interlab plates due to poor growth, allowed to grow over weekend (RC and AT).
Picked 2 colonies from each InterLab plate and grew over weekend (RC, AT and EM).

23/07/2018

Colony PCR for level 1 plates that haven’t shown bands yet.
PCR for Samples 13, 14, 15, 16a, 16b with 39 + 69 primers, ran gel - No bands except those in all samples - discussed possibility of water contamination/contamination in primers.
Made new plates and re-streaked colonies that haven’t yet worked from level 1 plates.
Grow overnight culture for the competent cells.
Level 0 digest for ATH and GUS.
Contacted iGEM HQ about collaborations.
Tutorial on how to use plate reader for interlab study
cPCR on InterLab plates (RC, AT, AND EM)

24/07/2018

Colony PCR for new streaked level 1 plates.
Re-run PCR for successful level 1 minipreps with new diluted primers 39+69.
Re-test level 1 colonies with primers 64+69 to double check before sending for sequencing.
Re-grew successful level 1 colonies.
Grow up overnight culture for competent cells in 800ml LB - cells didn’t grow properly so couldn't continue with competent cell protocol - instead set up a new overnight culture for the competent cells to start again tomorrow.
Transform G7 and 35S terminators from the iGEM registry (plate 6 14N + 16J) and then plate out and grow overnight.
1st Calibration completed for Interlab study, performed at 37°C.
Enhancer level 0 worked but GUS and ATH did not. Picked and grew enhancer overnight with good InterLab cells (RC and AT).
Set up level 0 dig/lig with GUS and ATH (RC and AT)

25/07/2018

Ran gel for yesterday’s PCRs.
Miniprepped colonies grown overnight and sent off for sequencing.
Tested all colonies from level 1 plates that haven’t yet worked with primers 64+69 and ran gel.
Make competent cells from the overnight culture.
Test the competent cells by doing a transformation using 18J, then plate out and grow overnight.
Colony PCR using the 14N and 16J colonies.
Run PCR products on a gel.
Grow up 14N and 16J colonies overnight.
Grow up GFP and 35S long from the glycerol stocks overnight.
2nd and 3rd Calibration completed, performed at 37°C.
Entered ILS data into excel (RC).
Performed culture part of ILS, took and measured t0 samples at 10:55, then t6 at 4:55. Measured Abs and Fluorescence (RC and EM).
Miniprepped enhancer (HE).

26/07/2018

Re-do level 1 reaction for 35S-BCR3_Full construct that has not yet worked - digestion and ligation overnight.
Miniprepped 14N, 16J, GFP and 35S from the overnight cultures.
Level 1 digest and overnight ligation using 35S/GFP/different terminators (Nos/G7/35S terminator).
Level 1 digest and overnight ligation using RTBVp/Enhancer/GFP/Nos.
Cell measurement protocol continued, dilution and measurements taken.
Colonies incubated overnight for Interlab study CFU section (RC and EM).
Entered ILS data and submitted measurement part (RC)

27/07/2018

Transformation of 35S-BCR3_Full construct and plating to grow over weekend.
Transformation of level 1 constructs (35S/GFP/terminors) using both bought and made competent cells to compare - plate out and grown over the weekend at 22℃.
Colony counting for Interlab study completed and Submitted to iGEM HQ. (RC and EM)
More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.
GUS and ATH level 0 dig/lig (RC and AT).

30/07/2018

Colony PCR - plates 1,2,3,4,5,6
Colony PCR - Lvl 1 constructs with different terminators - Ran on a gel-didn’t get bands.
Set up more colony PCRs for the white colonies overnight.
All GUS and ATH lvl 0 cells are blue (RC and AT)
Set up level 1 dig/lig reaction with RTBV, Enh, GFP, and NosT (REGT) and liagted overnight (RC and AT)

31/07/2018

Ran PCRs from yesterday on a gel - one colony had a band of the expected size so picked and grew that colony overnight.
PCR on more white colonies from the Lvl 1 plates (35s long/GFP/terminators).
Ran the PCR products on a gel - no bands.
Transformed cells with REGT. Ran one on a cycle and one separately to test (RC and AT).

01/08/2018

Level 1 transformation.
Grow up the 35s long and GFP overnight.
Miniprepped the 35S/GFP/35Sterminator colony that was grown overnight.
Miniprepped level 1 cultures grown overnight (14 and 16).
PCR on the miniprepped DNA (35S/GFP/35Sterminator and 14 and 16), the PCR products were then run on a gel.
cPCR or REGT constructs. All REGT modes worked, but cycle better so used from now on (RC and AT).
Grew more pSB1C3 overnight to do a diagnostic digest (RC)
Set up level 0 dig/lig with GUS and ATH (RC and AT)

02/08/2018

Sent for sequencing - 35SLong-GFP-35STerm with primer 69 and 35S-BCR3 with primer 64.
Blue/white selection on new transformed plates was poor so left in fridge overnight.
Miniprepped the 35S long and GFP grown overnight.
Set up new level 1 reactions for GFP with the various terminators.
Transformations using the level 1 reactions - plated onto Kan/IPTG and XGAL and grown overnight at 37℃.
Picked colonies from pSB1C3 into broth (RC)
Developed mathematical models (AT)

03/08/2018

Sent 35S-BCR3 for sequencing with primer 69.
Colony PCR for level 1 plates.
More seeds of Nicotiana benthamiana were planted and the condition of existing plants was monitored.
Colony PCR on the level 1 plates (GFP with various terminators) from yesterday - ran on a gel - one colony had a band of the expected size so that was picked and grown over the weekend.
Some of the plates didn’t have many colonies on so replated the rest of the transformation mixture from yesterday.
Minprepped REGT and pSB1C3 (RC and AT)
Performed diagnostic digest on pSB1C3 - BsmBI worked fine (RC and AT).
Set up lvl0 dig/lig for ATH as a cycle (RC and AT)

06/08/2018

Grew up all level 0 parts overnight.
Transformed GUS and ATH lvl 0 (RC and AT)
Miniprepped cultures for Emily (GFP terminator constructs) and set up PCRs to test. One was right so picked it and put into broth overnight (RC).

07/08/2018

Did level 1 reaction for 1-7 plates with NEB kit.
Miniprepped 35S long/GFP/G7 construct looked good so was sent for sequencing.
Redid the level 1 reactions (35S long/GFP/various terminators) with a new protocol.
Transformed the level 1 reactions and plated out on Kan/IPTG/XGAL and grew overnight at 37℃.
Developed navigation bar on the Wiki with help from WashU and their code (RC)
Colony PCR on GUS and ATH, but all too small (RC and AT).

08/08/2018

Ran colony PCR on new level 1 plates - gel wasn’t great (EH)
PCR run with 14,15,16,18, primers 64+70 and 64+71 were used - bands for 64+71 were strong (ST and HE)
Initial contact with WashU about potential collaboration via email and instagram (LT and EM)
Colony PCR on white colonies from the plates grown overnight. Ran the PCR products on a gel. Two colonies had bands around the expected size so were picked and grown overnight (EH)
Replated the rest of the level 1 transformation mix onto new plates (EH)
Sent REGT to be sequenced (RC)
Performed BsmBI digest on pSB1C3 alone and extracted the cut plasmid from the gel (RC).
Set up ligation reaction with cut pSB1C3 and GUS and ATH (RC and AT).

09/08/2018

Repeated colony PCR on new level 1 plates - gel slightly better so put successful colonies to grow overnight (EH)
Made new chloro broth (EH)
Grew up level 0 parts in correct broth (EH)
PCR run with 14,15,16,18, primers 69+70 and 69+71 were used. Bands for 69+70 were strong (ST and HE)
Video call with WashU detailing possible collaboration (all)
Miniprepped the two colonies grown overnight (EH)
Colony PCR on the white colonies from the level 1 plates (35S/GFP/terminators) and PCR on the miniprepped colonies (EH)
Ran the PCR products on the gel- only the miniprepped construct containing the G7 terminator had a band of the expected size (EH)
Transformed cells with GUS and ATH from the pre-digested plasmid (RC and AT)
Developed wiki front page and description (RC)
Developed models (AT)

10/08/2018

Sent the level 1 construct containing 35S long/GFP/G7 off for sequencing with primer 69 (EH)
Picked more white colonies off the terminator level 1 plates for PCR (EH)
Ran the PCR products on a gel - there were no bands of the expected size (EH)
Transformed Agrobacterium with REGT after it had its sequence confirmed. (RC and AT)
Ran cPCR on GUS and ATH, but negative had blue and white cells. Conclusion = the empty vector ligates to itself! (RC and AT).

13/08/2018

Sent the 35S long promoter off for sequencing with primer 57 (EH)
Set up a level 1 reaction using 35S short/Enhancer/GFP/Terminators (EH)
Transformation of the level 1 reaction - plate out onto Kan/IPTG/XGAL (EH)
Regrow the 35S long overnight (EH)
Had Esp3I delivered to replace BsmB1. Set up level 0 dig/lig with GUS and ATH (RC and AT).
One final cPCR on level 0 GUS cells with BsmBI used - tested 100 colonies - none with the correct size (RC and AT).

14/08/2018

Colony PCR on the white colonies from the level 1 reactions from yesterday (EH)
Run PCR products on a gel - most of the bands look good (EH)
Pick and grow the colonies overnight which had bands of the expected size on the gel (EH)
Miniprepped the 35S long which was grown overnight (EH)
Further discussion on logo design (EM and RC)
Transformed cells with GUS and ATH, which has Esp3I used (RC and AT).
Picked REGT Agro plates into kan/rif broth (RC and AT)

15/08/2018

Sent 35S long for sequencing with primer 68 (EH)
Miniprepped the cultures grown overnight (EH)
Sent one of each level 1 construct with different terminators off for sequencing (35S/enhancer/GFP/terminators) (EH)
Agro transformation with the level 1 constructs containing GFP and different terminators (EH)
Plants monitored and watered (EM)
Activated REGT agrobacterium, and also ST and HE lvl 1 constructs. Infiltrated plants (RC, AT and EM)
Ran cPCR on Esp3I level 0 white colonies (AT)
Several GUS level 0s worked so picked them into broth. One ATH looked promising so sequenced (RC and AT)
Set up another level 0 dig/lig with Esp3I, GUS and ATH (RC and AT)

16/08/2018

Grow seed overnight for making competent cells (EH)
Made up a solution of MgCl2 and autoclaved it (EH)
Obtaining contact information for potential collaborations (EM)
Miniprepped GUS and ATH, sequenced them (RC and AT)
Transformed cells with GUS and ATH level 0 constructs (RC and AT)
Set up level 1 reactions with GUS. 35S-Enh-GUS-NosT (3EGuT) and RTBV-Enh-GUS-NosT (REGuT) (RC and AT)

17/08/2018

Made competent E. coli (EH)
Transform new competent cells with 18J to test them - plate onto chloramphenicol and grow on the bench over the weekend. (EH)
Streaked out the agro plates and grow over the weekend at 28℃.
Sequencing came back for the 35S long, turned out it was 35S short which explains why the level 1s were not working.
Picked GUS and ATH white colonies and performed cPCR. Several GUS bands worked but no ATH bands (RC and AT).
Visualised REGT plants. Some results in the plant vasculature but very close to background levels (RC and ST)

20/08/2018

Pick colonies off the agro plates and grow cultures overnight in LB broth, Kan and Rif.
GFP and C002 plates had frozen at the back of the fridge, so repicked the colonies (RC).
Re-transformed 35S level 0, Enhancer, and pGB-A2 to miniprep more (RC).
Transformed 3EGuT and REGuT (RC and AT).
Made new Chl and Kan plates with fresh reagents (RC and AT).

21/08/2018

Infiltrated plants with 35S-GUS and either NosT, G7, or 35S (EH)
Re-potted plants (EM)
Coded the interactive cog buttons on the front page of the Wiki (RC).
cPCR on REGuT and 3EGuT. Most were good so picked into broth (RC and AT).
Picked Agro containing REGT constructs and 3EGT (35S-Enh-GFP-NosT) (RC and AT).
Picked Enh and grew in broth to miniprep more. 35S had not grown so set it up again. Picked pGB-A2 into broth (RC).
Picked good REGuT and 3EGuT cells into Kan broth (RC and AT).

22/08/2018

Level 1 Ligation for (15)cRTBVp_BCR3_Full and (18) 35S_BCR3_Full (HE)
Transformation of cells with Level 1s (HE)
Grew up four 11 35s (HE)
Transformed agro with REGuT and 3EGuT after miniprepping, and sequenced them (RC and AT).
Picked C12 ATH from 15/08/2018 and grew in broth (RC)
Infiltrated tobacco with REGT, 3EGT and -ve containing just pGB-A2 (RC, AT and EM).

23/08/2018

Preparation for Operation Earth. (LT)
Checked on progress of re-potted plants. (EM)
Miniprep of 35s (HE)
RNA Extraction from plants 14 and 16 (+ve and -ve) (ST)
Colony PCR for Plates 2 and 5 (HE)
Agro didn't grow enough to pick with GUS constructs so left for another day (RC and AT)
Miniprepped level 0 ATH (AT)

24/08/2018

RT Reaction (-ve, 14, 16) (ST)
Picked GUS level 1s from plates and streaked onto new plates (RC and AT).
Sent level 0 ATH for sequencing (RC and AT).
Soldered the plant light's wiring and circuit. Sanded parts to fit and glued them within the case. Tested the light and learnt that 1 LED does't survive the current from 8 AAA batteries. Finally left at 8pm (RC and AT).
Bought more LEDs for the leaf 3D model (LT)

28/08/2018

Tested new primers with 14 and 16 (ST)
cPCR on other ATH colonies - no good bands (RC and AT)
Ribitol control element gBlock came so set up level 0 reaction and transformed (RC and AT).
Set up lvl 0 reaction with ATH again (RC and AT).
Picked GUS lvl 1 constructs into Rif/Kan broth (RC and AT).

29/08/2018

PCR with cDNA + gDNA (ST)
mCherry gBlock arrived.
cPCR on ribitol control element (RCE) and ATH level 0s - no good bands (RC and AT).
Transformed plants with GUS lvl 1s but MgCl₂ and MES concentrations were halved by a calculation error (RC and AT)
Set up level 0 digest ligation with RCE, ATH and mCherry (RC and AT).

30/08/2018

Transformed lvl 0 RCE, ATH and mCherry and plated them (RC and AT)

31/08/2018

cPCR on lvl 0 ATH, RCE and mCherry (RC and AT).
RCE and mCherry had good bands so picked them and grew over weekend (RC and AT).
Grew 3EGuT and REGuT in broth too (RC and AT).
Met with Dr. Pass for bioinformatics - he gave us the script and terminal to use (RC and AT).

03/09/2018

Miniprepped and sequenced RCE and mCherry (RC and AT).
Performed GUS assay on 3EGuT and REGuT (RC and AT)
Infiltrated plants with 3EGuT and REGuT again (RC and AT)
Set up lvl 1 reaction with 35S-Enh-mCherry-NosT (3ECT), RTBV-Enh-mCherry-NosT (RECT), RCE-Enh-mCherry-NosT (RiECT), and RCE-Enh-GUS-NosT (RiEGuT) (RC).

04/09/2018

Transformed and plated 3ECT, RECT, RiECT, and RiEGuT (RT and AT).
Miniprepped more RCE and mCherry (AT).
Destained GUS constructs but there was no GUS activity in the first REGuT and 3EGuT leaves (with half concentration activation buffer) (RC).

05/09/2018

cPCR on level 1 3ECT, RECT, RiECT, and RiEGuT. All 35S and RTBV worked, but no RCE worked (RC and AT).
Heat shocked plants with REGuT and 3EGuT (RC and AT).
Set up lvl 0 RCE reaction again (RC)

06/09/2018

Transformed E. coli with RCE level 0s (RC).
Transformed Agro with 3ECT and RECT after miniprepping (RC and AT).
Sequenced mCherry, 3ECT, RECT and 3EGuT after miniprepping (RC and AT).

07/09/2018

mCherry level 1s didn't grow enough so left over weekend (RC).
cPCR on RCE, some worked so picked into broth (RC and AT).
Re-picked ReGUT and 3EGuT into broth (RC and AT).
Added Dr. Scofields GUS staining solution to previous ReGUT and 3EGuT solutions and left at 37 degrees Celsius over weekend (RC).

10/09/2018

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11/09/2018

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12/09/2018

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13/09/2018

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14/09/2018

Last official day so cleaned up the lab (all)

17/09/2018

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18/09/2018

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19/09/2018

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20/09/2018

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21/09/2018

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21/09/2018

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24/09/2018

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24/09/2018

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25/09/2018

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26/09/2018

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27/09/2018

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28/09/2018

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31/10/2018

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