Difference between revisions of "Team:Cardiff Wales/Notebook/Text"

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<p>
 
<p>
 
<ul type="circle">
 
<ul type="circle">
−
<li>Miniprepped the WRKY intron cultures grown over the weekend. </li>
+
<li>Miniprepped the WRKY intron cultures grown over the weekend. (EH) </li>
 
<li>Make plates containing Chloro/XGAL/IPTG and Kan/XGAL/IPTG. </li>
 
<li>Make plates containing Chloro/XGAL/IPTG and Kan/XGAL/IPTG. </li>
 
<li>iGEM white check in form for using aphids (Health and Safety). </li>
 
<li>iGEM white check in form for using aphids (Health and Safety). </li>
Line 108: Line 108:
 
<li>Set up Lvl 1 reactions for successful Level 0 constructs (HE and ST). </li>
 
<li>Set up Lvl 1 reactions for successful Level 0 constructs (HE and ST). </li>
 
<li>Contacted Thea (European iGEM ambassador) about collaborations.</li>
 
<li>Contacted Thea (European iGEM ambassador) about collaborations.</li>
−
<li>Colony PCR for Lvl 0 colonies (Enhancer, 7 and 10).</li>
+
<li>Colony PCR for Lvl 0 colonies (Enhancer, 7 and 10). (EH) </li>
−
<li>Run PCR products on a gel - there were no bands.</li>
+
<li>Run PCR products on a gel - there were no bands. (EH) </li>
 
<li>Discussion about logo design.</li>
 
<li>Discussion about logo design.</li>
 
<li>GUS, ATH and Enhancer didn't work, so re-performed Dig/Lig (RC and AT).</li>
 
<li>GUS, ATH and Enhancer didn't work, so re-performed Dig/Lig (RC and AT).</li>
Line 122: Line 122:
 
<li>Contact Fiona about risk assessment for human outreach event at Techniquest.</li>
 
<li>Contact Fiona about risk assessment for human outreach event at Techniquest.</li>
 
<li>Meeting with Dan to discuss 3D printing for human outreach. </li>
 
<li>Meeting with Dan to discuss 3D printing for human outreach. </li>
−
<li>Transformations using the iGEM registry parts for the interlab study. </li>
+
<li>Transformations using the iGEM registry parts for the interlab study. (EH) </li>
 
<li>GUS,ATH, Enh cells grew but were all white. Performed colony PCR (cPCR) anyway but none worked. (RC and AT).</li>
 
<li>GUS,ATH, Enh cells grew but were all white. Performed colony PCR (cPCR) anyway but none worked. (RC and AT).</li>
 
<li>Made fresh kanamycin broth (HE and ST).</li>
 
<li>Made fresh kanamycin broth (HE and ST).</li>
Line 162: Line 162:
 
<li>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 (HE and ST).</li>
 
<li>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 (HE and ST).</li>
 
<li>Made new plates and re-streaked colonies that haven’t yet worked from level 1 plates (HE and ST).</li>
 
<li>Made new plates and re-streaked colonies that haven’t yet worked from level 1 plates (HE and ST).</li>
−
<li>Grow overnight culture for the competent cells.</li>
+
<li>Grow overnight culture for the competent cells. (EH) </li>
−
<li>Level 0 digest for ATH and GUS.</li>
+
<li>Level 0 digest for ATH and GUS. (EH) </li>
 
<li>Contacted iGEM HQ about collaborations.</li>
 
<li>Contacted iGEM HQ about collaborations.</li>
 
<li>Tutorial on how to use plate reader for interlab study</li>
 
<li>Tutorial on how to use plate reader for interlab study</li>
Line 180: Line 180:
 
<li>Re-test level 1 colonies with primers 64+69 to double check before sending for sequencing (HE and ST).</li>
 
<li>Re-test level 1 colonies with primers 64+69 to double check before sending for sequencing (HE and ST).</li>
 
<li>Re-grew successful level 1 colonies (HE and ST).</li>
 
<li>Re-grew successful level 1 colonies (HE and ST).</li>
−
<li>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. </li>
+
<li>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. (EH) </li>
−
<li>Transform G7 and 35S terminators from the iGEM registry (plate 6 14N + 16J) and then plate out and grow overnight.</li>
+
<li>Transform G7 and 35S terminators from the iGEM registry (plate 6 14N + 16J) and then plate out and grow overnight. (EH)</li>
 
<li>1st Calibration completed for Interlab study, performed at 37°C.</li>
 
<li>1st Calibration completed for Interlab study, performed at 37°C.</li>
 
<li>Enhancer level 0 worked but GUS and ATH did not. Picked and grew enhancer overnight with good InterLab cells (RC and AT). </li>
 
<li>Enhancer level 0 worked but GUS and ATH did not. Picked and grew enhancer overnight with good InterLab cells (RC and AT). </li>
Line 196: Line 196:
 
<li>Miniprepped colonies grown overnight and sent off for sequencing (HE and ST).</li>
 
<li>Miniprepped colonies grown overnight and sent off for sequencing (HE and ST).</li>
 
<li>Tested all colonies from level 1 plates that haven’t yet worked with primers 64+69 and ran gel.</li>
 
<li>Tested all colonies from level 1 plates that haven’t yet worked with primers 64+69 and ran gel.</li>
−
<li>Make competent cells from the overnight culture. </li>
+
<li>Made competent cells from the overnight culture. (EH) </li>
−
<li>Test the competent cells by doing a transformation using 18J, then plate out and grow overnight.</li>
+
<li>Test the competent cells by doing a transformation using 18J, then plate out and grow overnight. (EH) </li>
−
<li>Colony PCR using the 14N and 16J colonies.</li>
+
<li>Colony PCR using the 14N and 16J colonies. (EH) </li>
−
<li>Run PCR products on a gel. </li>
+
<li>Run PCR products on a gel. (EH) </li>
−
<li>Grow up 14N and 16J colonies overnight. </li>
+
<li>Grow up 14N and 16J colonies overnight. (EH) </li>
−
<li>Grow up GFP and 35S long from the glycerol stocks overnight.</li>
+
<li>Grow up GFP and 35S long from the glycerol stocks overnight. (EH) </li>
 
<li>2nd and 3rd Calibration completed, performed at 37°C. </li>
 
<li>2nd and 3rd Calibration completed, performed at 37°C. </li>
 
<li> Entered ILS data into excel (RC). </li>
 
<li> Entered ILS data into excel (RC). </li>

Revision as of 20:20, 4 October 2018


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. (all)
  • 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 (HE and ST)
  • 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℃. (EH)
  • 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. (EH)
  • 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 (HE and ST)
  • Sent off Lvl 0 constructs for sequencing (HE and ST).
  • 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 (HE and ST).
  • Contacted Thea (European iGEM ambassador) about collaborations.
  • Colony PCR for Lvl 0 colonies (Enhancer, 7 and 10). (EH)
  • Run PCR products on a gel - there were no bands. (EH)
  • 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 (HE and ST).
  • 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. (EH)
  • GUS,ATH, Enh cells grew but were all white. Performed colony PCR (cPCR) anyway but none worked. (RC and AT).
  • Made fresh kanamycin broth (HE and ST).






19/07/2018

  • Colony PCR for new lvl 1 plates with primers 64+69 and gel run (HE and ST).
  • Colonies with correct band sizes grown overnight (HE and ST).
  • 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 (HE and ST).
  • Colony PCR for level 1 plates that didn’t show bands in yesterday’s colony PCR (HE and ST).
  • 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 (HE and ST).
  • 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 (HE and ST).
  • Made new plates and re-streaked colonies that haven’t yet worked from level 1 plates (HE and ST).
  • Grow overnight culture for the competent cells. (EH)
  • Level 0 digest for ATH and GUS. (EH)
  • 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 (HE and ST).
  • Re-run PCR for successful level 1 minipreps with new diluted primers 39+69 (HE and ST).
  • Re-test level 1 colonies with primers 64+69 to double check before sending for sequencing (HE and ST).
  • Re-grew successful level 1 colonies (HE and ST).
  • 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. (EH)
  • Transform G7 and 35S terminators from the iGEM registry (plate 6 14N + 16J) and then plate out and grow overnight. (EH)
  • 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 (HE and ST).
  • Miniprepped colonies grown overnight and sent off for sequencing (HE and ST).
  • Tested all colonies from level 1 plates that haven’t yet worked with primers 64+69 and ran gel.
  • Made competent cells from the overnight culture. (EH)
  • Test the competent cells by doing a transformation using 18J, then plate out and grow overnight. (EH)
  • Colony PCR using the 14N and 16J colonies. (EH)
  • Run PCR products on a gel. (EH)
  • Grow up 14N and 16J colonies overnight. (EH)
  • Grow up GFP and 35S long from the glycerol stocks overnight. (EH)
  • 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 (HE and ST).
  • 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 (HE and ST).
  • 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, colonies with bands at the expected size left to grow in broth overnight (HE and ST).
  • Concern that some of the colonies picked up with a tip may actually be two small colonies combined, so single colonies from lvl 1 plates streaked on a fresh plate to ensure single colonies and left to grow overnight (HE and ST).
  • 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

  • Colony PCR for fresh streaked plates, colonies with a band at the expected size were grown overnight (HE and ST).
  • Miniprepped colonies grown overnight.
  • 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.
  • New lvl 1 reaction for all constructs (HE and ST).
  • Transformed cells with REGT. Ran one on a cycle and one separately to test (RC and AT).






01/08/2018

  • Level 1 transformation (HE and ST).
  • 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, HE and ST).
  • 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 (EH and HE).
  • Blue/white selection on new transformed plates was poor so left in fridge overnight (HE and ST).
  • 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 (HE and ST).
  • Colony PCR for level 1 plates, successful colonies from plates 1, 3, 5 and 6 were left to grow over the weekend (HE and ST).
  • 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

  • Put all level 0 parts in broth to grow overnight (HE and ST).
  • 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 (HE and ST).
  • 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).
  • Lvl 0's grown overnight were accidentally grown in wrong broth so level 0's were grown in correct broth overnight (HE).






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), along with lvl 1's (ST and HE).
  • 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)
  • Transformed Agrobacterium with successful lvl 1s (HE and ST).
  • 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).
  • Streaked Agrobacterium plates that had grown over the weekend onto a kan/rif fresh plate (HE and ST).






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)
  • Picked successful lvl 1 Agro plates into kan/rif broth (HE and ST).






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)
  • Infiltrated plants with lvl 1 Agrobacterium (HE and ST).






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)
  • More colony PCR from lvl 1 plates we didn't have a successful construct for (HE and ST).






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)
  • Colony PCR on lvl 1 plates - gel accidentally run without markers so results inconclusive (HE).






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).
  • Re-ran last week's lvl 1 PCR on a gel - with markers this time (HE).
  • Lvl 1 reaction for SP3_5 constructs (HE and ST).






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)
  • Colony PCR for plate 5 (HE).






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).
  • Re-picked Agro colonies from successful lvl 1 plates and put into kan/rif broth for infiltration.






10/09/2018

  • Streaked mCherry constructs and picked into broth (RC).
  • Destained GUS constructs in ethanol - GUS worked this time with Dr. Scofield's reagents (RC).
  • Activated and infiltrated 3EGuT and REGuT again (RC).
  • Miniprepped RCE lvl 0 and set up lvl 1 reaction with it and mCherry or GUS (RC).
  • Activated and infiltrated Emily, Sophie and Hannahs cells as they were away (RC, LT and AT).






11/09/2018

  • Set up level 0 reaction with full composite mCherry construct to test in bacteria, as it arrived as a gBlock (RC).
  • Activated and infiltrated mCherry constructs. This time to heat shock 1DPI and test 3DPI (RC).
  • Sent RCE off for sequencing and some of Sophie/Hannah's constructs (RC).
  • Transformed E. coli with RCE level 0s (AT).
  • Destrained Emily's GUS terminator assays, and found G7 was strongest (RC).






12/09/2018

  • cPCR on RCE lvl 1 constructs. All bands are the correct size, so picked one RiECT and one RiEGuT (RC and AT).
  • Transformed E. coli with bacterial mCherry composite part (RC).






13/09/2018

  • Miniprepped and transformed Agro with RiEGuT and RiECT (RC).
  • Visualised mCherry in bacteria with Jamie - strong expression (RC).
  • Heat shocked final REGuT and 3EGuT plants (2DPI) and mCherry plants (1DPI) (RC).
  • RNA extraction from infiltrated plants (HE and ST) and cDNA prep (ST).






14/09/2018

  • Started GUS assay on 3EGuT and REGuT (RC).
  • Visualised mCherry in plants with Jamie - strong expression, mostly in 3ECT constructs (RC).
  • Last official day so cleaned up the lab (all)
  • PCR with cDNA (ST).






17/09/2018

  • Streaked RiEGuT and RiECT and picked into broth (RC).
  • Destained 3/REGuT leaves (RC)
  • Picked bacterial mCherry into broth (RC).






18/09/2018

  • Realised there was a miscommunication about what RCE is. Its part of a Ribitiol operon and isn't just a promoter, but a repressor as well. Decided to carry on with the Collaboration and testing anyway, as the results could be unusual (RC).
  • Grew up all constructs for glycerol stocks and dry DNA send-off (RC).
  • Skyped Kyle from WashU at midnight to discuss 3D model, testing the constructs, and documenting the collaboration (RC)






19/09/2018

  • Miniprepped all lvl 0s and stored in Freezer for Geraint to send off on the 08/09/2018 (RC).
  • Made glycerol stocks of all parts (lvl 0 and lvl 1) (RC).







21/09/2018

  • Grew agro over the weekend to infiltrate on the Monday. Watered plants (RC).







24/09/2018

  • Activated and infiltrated RiEGuT and RiECT (RC).
  • Sequenced Bacterial mCherry (RC).
  • Miniprepped and made glycerol stock of RCE lvl 0 (RC).






26/09/2018

  • Heat shocked RiEGuT and RiECT plants (RC).






27/09/2018

  • Infiltrated ribitiol into plants at varying concentrations. Leaf 1 was 10mM, leaf 2 5mM, leaf 3 1mM, and leaf 4 not activated. Did not treat the negative control (RC).







28/09/2018

  • Performed GUS staining assay to destain on Monday (RC).
  • Visualised RiECT in the plants. Unusual results. There was no expression around the leaf, but had high expression levels at sites of infiltration, where the Agrobacterium are most concentrated. Activation with ribitol did not make a difference (RC).







01/10/2018

  • Destained RiGuT constructs (RC).







31/10/2018

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