Team:Cambridge/Notebook/Week11

Monday

Theo made a video of the bacterial bubble lamp, set up in the lab. He also attempted to ligate pBAD to EPIC luciferase, which had so far failed. He was pleased to find that he managed to change the colour of LC luciferase to green in a previous experiment, but had only a single colony so plated this out again.

Tuesday

To our surprise the EPIC pBAD was reddish, we made up a culture for sequencing to check that it was indeed EPIC, Theo performed a colony PCR which seemed to suggest it was. Ben and then Theo attempted to change the colour of LC luciferase from wildtype. Theo got good results for the attempt to change the 433rd amino acid, and he performed Gibson assembly and transformation.

Wednesday

Theo retransformed his Gibson reaction because of contaminated SOC media. Paul and Peter put on a plate reader experiment to test the uptake of luciferin at a range of pHs. Emily sent DNA for sequencing and started biobrick assembly of pBAD with the separated luciferase and LREs from DNA2.0, as well as Theo's EPIC luciferase with LRE.

Thursday

Our sequencing results today were very exciting, because they showed that our attempt to separate the luciferases from LREs from DNA2.0 had been successful! Hooray! Emily continued with biobrick assembly to put these and EPIC luciferase+LRE under a promoter (after running out of pBAD and forgetting to restrict some things yesterday). Peter was doing excellent t-shirt design and Ben continued looking into some human practises stuff. Paul continued with modelling and analysing results from the plate reader.

Friday

Peter was in communication with the Mexico UNAM Genomics iGEM team today because he was sending the DNA from most of our light producing parts we'd made over the summer. Mexico needed some working light output to test their communication project and so we were happy to help. After the success of the week's biobrick assembly Emily continued in her quest to finish typing up the lab book.

And of course, it was dress-up-Friday. Theo made sure to make the effort this week.

Saturday

Sunday

Monday

Tuesday

107. Expt: Biobrick assembly of pBAD+Luc (PP + LC) and (Emily)

• Plasmids already extracted
• Restriction digest following protocol on p88 using these quantities (in µl):

• Gel electrophoresis
  • pBAD failed - band at ~5000bp, should be 1200
  • others look about right ~ between 3 & 5kb (should be 3600bp)

• Gel extraction - results in -20°C freezer

Wednesday

108. Expt: Send off PP + pSB1C3, EPIC pBAD for sequencing (Emily and Peter)

• Miniprep EPIC pBAD and nanodrop
• Prepare correct concentrations (100ng/µl for plasmid, 3.2pmol/µl for primers) to be sent off

109. Expt: Biobrick assembly of pBAD + PP Luc (1), PP Luc (2), LC Luc (1), PP+pSB1C3

• Peter 'miniprepped' PP+pSB1C3 to extract plasmid
• pBAD was amplified after trying to grow overnight culture failed. The following protocol was used:

Add to a PCR tube:

• Use stab to extract colony from pBAD plate and swirl in tube. Run following program on PCR machine saved as 'pBAD Amplification':
  • Heated lid at 110°C
  • Denaturation for 1m30s at 98°C
  • Cycle 35 times
    • Denaturation for 30s at 98°C
    • Elongation for 2m at 72°C
  • Elongation for 7m30s at 72°C

• PCR Purification of pBAD from PCR reaction was performed using Qiagen kit
• Restriction: repeating restriction of LC Luc (1) because tube from yesterday was dropped. Use protocol on p88 with following quantities (in µl):

'*' Nanodrop said 29ng/µl after PCR Purification, which was strangely low. So used 16µl as recommended (in order to not exceed 0.2µg) but maximise amount of DNA.

• Gel electrophoresis: Run gel with following quantities:

110. Expt: Plate reading experiment to investigate effect of pH on light output

3pH: 5.3, 6.1, 7

D-luciferin and Caged D-luciferin (at 10mM mDMSO)

Plate layout:

• Arabinose: 100µM
• Luciferin: 100µM

• 3 wells with Arabinose only, and 3 blank wells. Buffer at 7.
• 100µl per well
• 50µl of cells
• 48.5µl of Buffer
• 1.5µl of Luc/1µl of caged Luc
• 1µl of Arabinose

111. Expt: continuation of Biobrick assembly of pBAD with P.P.Luc(1), P.P.Luc(2), L.C.Luc(1), P.P.pSB1C3 (Emily)

• Ligation: Accidentally forgot to restrict P.P.pSB1C3 so will do that later. Followed ligation protocol on p91 with following quantities:

Cells that were ligated were then transformed

There was not enough pBAD to ligate L.C.Luc(1) ==> PCR lots of pBAD.

pBAD Colony PCR

Mix in 3x PCR tubes:

Run program from p101.

• PCR Purification: using Qiagen kit
• Nanodrop:
  • Tube 1 --> 36.7ng/µl
  • Tube 2 --> 69.1ng/µl
  • Tube 3 --> 66.2ng/µl
• Restriction: Following protocol on p88 with these quantities:

• Ran on a gel with 17µl DNA + 3µl of 6x orange LD in this order: Hyperladder I, pBAD 1, pBAD 2, pBAD 3, PP Luc2, PP pSB1C3
• All bands were as expected: pBAD - 1200bp, PP Luc(2) - 3600bp, PP pSB1C3 - ~4500bp
• Ben gel extracted

Ligation

Following protocol on p91, incubating at RT for 30 mins. The nanodrop reading for PPLuc(2) was so bad (2.8ng/µl) that the ligation was not performed because PPLuc(1) had already been transformed with a small amount of pBAD yesterday. Wait for results of that.

Nanodrops:

• pBAD 1 -> 3.2ng/µl
• pBAD 2 -> 34.5ng/µl
• pBAD 3 -> 15.3ng/µl
• PP pSB1C3 -> 11.5ng/µl
• LC Luc(1) -> 53.2ng/µl

Cells were then transformed.

Results

• Glowing growth on PPLuc(2) plates and PP pSB1C3 plates
• Growth but no glow on LC Luc(1) plates
• No growth on PP Luc(1) plates

Results later that day:

• LC Luc(1) now glowing :)

Thursday

112. Expt: Plate reader experiment. G28 and effect of D-luciferin on LC+LRE

This experiment was designed to measure the effect of Arabinose on G28 light output and D-luc level on LC/Red mutant. The layout was as follows (Blanks contain 30µl H20 + 70µl LB):

• B-D: Arabinose level varied in G28
• E-G: D-luc level varied in LC (red)
• 3 times repeats were made of the concentration given above. Total cell volume was 70µl for G28, 84µl for LC (red) and a total of 100µl per well.

Friday

113. Expt: Cultures for Mexico (Peter)

Grew up liquid culture of:

• 1. G28
• 2. PP Luc + LRE
• 3. pBAD + PP Luc + LRE
• 4. LC Luc + LRE
• 5. pBAD + LC Luc + LRE

Saturday

Plasmid miniprepped all 5 cultures. Nanodrop results:

in 50µl of EB.

Placed cryo tubes in freezer