Week 2

19/07/2015

• Prepared 2 LB Broth agar bottles, 1 with Kanamycin and 1 with Ampicillin antibiotic.
• We switched backbones for the promoter and terminator, both to Kanamycin from Chloramphenicol. The protocol for digestion and ligation is outlined below:

Preparation of the agar for plates:

• 10g of agar powder in 500ml of water in each bottle.
• Autoclave the bottles (1 hour and 30mins)
• For the antibiotic stock solutions:
  1. Prepared 50mg/ml of stock solution of Kanamycin antibiotic. Dissolved Kanamycin powder in water (not ethanol, as Kanamycin is soluble in water)
  2. For Ampicillin, prepared a stock solution of 100mg/mL, in water.
• Added 500ul of ampicillin to the LB agar and 1ml of Kanamycin into the other LB agar bottle as Kanamycin is less potent than ampicillin. Final concentration of ampicillin and Kanamycin was 50ug/mL
• Once cooled, pour into plates under the fume hood.
  Labelling:
  1. line for Ampicillin
  2. lines for Kanamycin
  3. lines for Chloramphenicol

Ligation and Digestion:

* The restriction enzymes we used were EcoRI and Pst1.

1. Add 38ul of water to each of the tubes
2. Add 5ul of plasmids. The tubes we used were PR2 (for promoter and RBS), and TR1(terminator). These tubes had the best results for the Nanodrop.
3. Add 5ul of NEB Buffer 2.1 to all the tubes.
4. Spin at 8000 rpm for 30 seconds.
5. Then add 1ul of restriction enzymes EcoRI and Pst1 to the tubes.
6. Spin and incubate at 37˚C for 15mins.
7. Inactivate the enzymes by incubating them at 80˚C for 20mins.
8. For the ligation, we add 7ul of H20 ( DNAase and RNAase free) to clean, labelled Eppendorf tubes.
9. Add 2ul of ligase reaction buffer (T4 DNA ligase buffer)
10. Then add 5ul of the digest into each tube.
11. Add 1ul of T4 DNA ligase.
12. Spin the tubes in the centrifuge for 30seconds at 13,000rpm.
13. Incubate the mixture for 30mins at 16˚C.
14. Inactivate the enzyme by incubating the mix at 65˚C for 10mins, as to prevent over ligation

All of the digest, antibiotics and buffers were placed in the deep freezer ( -38˚C). The spare agar plates were placed in the fridge, both Kan. and Amp.

Transformation and agar culture:

1. 5ul of ligated mixture used for each tube of competent cells.
2. Place the tubes on ice for 30 minutes
3. Heat shock at 42˚C for 40 seconds
4. Pre-warm the plates at 37˚C.
5. Streak 2 plates with SOC recovery culture at original concentration
6. Streak 2 plates with culture diluted with 300ul SOC.
7. 100ul of culture was spread on each plate.
8. 4plates (as mentioned in the 2 above steps) were incubated at 37˚C in the incubator at 1841h.

20/07/2015

• 1046h: The agar plates that were inoculated yesterday show slight growth. Multiple colonies were observed but the average colony size is small. The plates were left in the incubator so the colonies can growth further.
• The control plate was prepared with TR1 on Kanamycin backbone using LB+Kan plate. The control plate was placed in the 37˚C incubator at 1111h. Plated from strong liquid culture so growth expected rapidly if plate antibiotic was impotent.
• LB Broth was prepared with 12.5g of L1325 from Sigma ( LB Broth powder) in 300ml of water. It was placed in the autoclave at 1113h.
• LB broth was removed from the autoclave and cooled at 1241h.
• The control plate was prepared on LB+ Amp. plates with TR1 on CamR backbone. This was incubated at 37˚C at 1204h.
• No red colonies or fluorescence were observed on any plates under UV light after 7hours. Plates were left in incubator.

21/07/2015

• All the cells that grew are white(no red cells). Hence, today we will transform the RFP plasmids to see if there is growth. If not, then there is a mistake. We will do 2 transformations of 2 separate RFP plasmids- Amna’s and iGEM’s and two separate antibiotics (Ampicillin + Kanamycin)
• We discovered that a mistake was made with the RFP cells, and they were GFP plasmids. So we will re-do the digestion and ligation.
• We redigested the initial plasmids- the promoter + terminator, so we can switch backbones.
• Repeated the digestion and ligation process ( see above for protocol)
• Did the transformation process
  • Used 950ul SOC and shaking it at 250rpm for 1 hour. Greater shaking leads to better growth, it seems.
• We also ordered TnaA on Ampicillin from IDT, and TnaB on Kanamycin from IDT.
• Incubated the cells in the 37˚C incubator overnight. It was placed in the incubator at 1807h. We did not dilute them in SOC medium, instead plating 100ul of each cells on agar plates (2 Kan. plates and 2 Amp. plates)

22/07/2015

• 1122h: Agar plates checked for growth-there was good growth with distinct colonies on all plates Red fluorescence observed under UV for some colonies.
• 1125h: Agar plates placed back in the incubator.
• 1600h: Reddish colonies were observed, so we are waiting for clearer difference in colour in the cells.

Liquid media calculations:

Kan:
Final conc: 50ug/mL
Final vol.: 10mL
Stock conc: 50mg/mL


Dilution: 10ul of stock solution + 10ml of LB Broth

Amp:
Final conc: 100ug/mL
Final vol.: 10ml
Stock conc.: 100mg/ml

Dilution: 10ul of stock solution + 10ml of LB Broth

Inoculation: The colonies were picked from the plates. They were not as close to differentiating between red and white but we will still inoculate in tubes with 10mL of LB Broth tubes, for each of PR2 and TR1 (Amp. + Kan. both respectively). Hence, it may be red!
Inoculated at 2200h. We also created duplicates of the LB Broth tubes and picked duplicate colonies of the plates because of the uncertainty regarding the colour of colonies. We followed the same standard procedure as outlined above (10ul of each antibiotic in 10ml of broth)
The tubes were then inoculated in the incubator, shaken at 250rpm horizontally.

23/07/2015

• The cells grew, at least 1 tube per term and promoter, 4 tubes had no growth.
• We decided to wait until the evening to complete the inoculation.
• Performed triplicates of the inoculation, following standard protocols for inoculation as outlined in previous weeks.

24/07/2015

• Visible growth for all terminator tubes
• Upon centrifuging the tubes, white residue was observed- indicating white cells. Hence we performed miniprep according to standard protocols
• There was not visible growth for all promoter tubes. However, upon centrifuging, there was some residue observed and so we resuspended the pellet and allow to grow further
• The miniprep for terminator tubes was performed according to standard protocol, with the exception that we did not add LyseBlue reagent to the tubes.

ligation

• 5ul of terminator digests and chitinase digests. ( 5ul terminator and 5ul ChA +5ul backbone)
• 2ul of DNA ligase was added ( double the amount)
• 2ul of water was also added to the tube( DNA and RNAse free water)
• at 1755h, growth observed in all 3 tubes containing promoters on KanR backbones. It was spun down in the centrifuge at 4000rpm for 3 mins. All pellets appear white.
• The Miniprep carried out on all three 3 tubes separately to avoid cross contamination. The Miniprep was done according to standard protocol.
• Transformation of the ChiA- terminator was also done today.
• We also made new agar plates with Ampicillin antibiotic
• Plasmids were then placed in the fridge.

Agar Plates

1. Use the antibiotic of ampicillin stock concentration of 100mg/mL
2. Place 35g of agar powder in 1L of water then autoclave the bottle.
3. Added 1ml of antibiotic to the bottle, then pour out into plates under fume hood.

Transformation of ChiA- terminator construct:

1. Use the standard ligation protocol, using 5ul of ligation mixture used for each 2 tube of competent cells.
2. 3 ligation replicates transformed separately into separate tubes of cells.

25/07/2015

• Saw good growth on the plates, that is, distinct white colonies on the plates (this is the terminator + chitinase construct that was transformed yesterday) so we picked single colonies and inoculated them in LB
  10ml of LB Broth and 10ul of ampicillin( with a stock concentration of 100mg/mL)
• They were placed in the shaking incubator at 250rpm horizontal shaking at 37˚C at 2022h.