Exterminator Coli

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Wet Lab

Week 1:

15/07/2015

We started the miniprep using the instructions of QIAprep Spin Miniprep Kit:

• Add RNase A solution (200 ul of concentration mg/ul) to Buffer PI (20ml) and mix, store at 2-8˚C. This was stored in the fridge, 3rd from right. (We did not add the LyseBlue reagent to the Buffer PI )
• Centrifuge the overnight culture (1ml in each tube) of 8200rpm for 3mins at room temperature
• Pour out the LB, so that the pellet remains, then resuspend the pellet in 250 ug of Buffer solution. Then transfer to a microcentrifuge tube.
• Add the 250ug of P2, mix thoroughly. Do not allow to proceed for more than 5 mins.
• Add 350ug of Buffer N3 , mix immediately, invert 4-6 times.
• Centrifuge for 13,000 rpm for 10mins in the centrifuge
• Apply the supernatant, centrifuge for 30-60secs, discard flow through
• Add 500ul pf PB buffer to wash and centrifuge for 30-60seconds. This was an optional step that was done in our process
• Wash QIAprep spin column, add 750ul Buffer PE. Centrifuge for 30-60seconds. (We prepared glycerol stock of liquid culture, with equal parts of glycerol and cell culture of 500ul. This was stored in the -8˚C fridge.)
• Centrifuge for 1 min to remove residual wash buffer.
• Place QIAprep column in a clean eppendorf tube. Add 50ul of water ( not the Buffer EB), let it stand for 1 min, then centrifuge for 1 minute.

The DNA was stored in the fridge at 4 ˚C overnight.

Note: We used deionized water for step 10, so we are not sure if it is DNA free and RNA free. So we have to see if there is any degradation tomorrow.

16/07/2015

We performed a Nano drop to check the quality of DNA. We pipetted 1ul of DNA into the machine.

Results

-tr1, tr2, BBA006, BBA700 are of poor quality so we will grow and miniprep them again. With picking a colony, we take 250ul of LB then incubate for hours, then plate.

1. Use the culture from 12/7/15. (iGEM 2015 plates were from 13/07)
2. 250ul of LB Broth is added to each of the picked colonies and they go in the shaking incubator for several hours ( 2-3 hours), shaking at 180rpm. * colonies were picked from the plate of 006 and 700 cells, as well as from original agar stabs from iGEM.
3. Re-doing the miniprep for the rest of the tubes and we used the rest of the liquid culture to get a better result in the Nanodrop.
4. Follow QiaPrep Spin Miniprep Kit protocol, but this time we added LyseBlue to the mixture, perhaps there was too little of solution because it did not turn blue but we could see the precipitate (i.e. cloudy, but colourless solution)
5. Skipped Step 7 of the protocol as it was optional, i.e. we did not add Buffer PB.

* Instead of deionised water, we added Buffer EB to elute

RESULTS OF 2nd Nano drop:

Results Of 2nd Nano Drop

Tube	Nucleic acid ng/ul	A260	A280	260/280	260/360
PR1	99.3	1.986	1.052	1.89	1.77
PR2	75.6	1.511	0.787	1.92	1.64
TR1	48.6	0.973	0.491	1.98	1.67
TR2	63.1	1.262	0.653	1.93	1.82
PR5	37.5	0.749	0.401	1.87	0.98
BBA006	49.0	0.981	0.510	1.92	1.46

15/07/2015

• The plated cells from liquid culture (16/07/15 ) that were inoculated from 250ul liquid cultures show good growth. These plates were raised from agar stabs shipped from iGEM.
• We picked single colonies from each plate and inoculated in LB and Chloroamphenicol liquid medium. Each colony picked was placed in 10ml of L.B. liquid medium in each tube. The inoculation occurred under sterile conditions i.e. under the fume hood with a Bunsen burner.
• The plates were stored in 4˚C refrigerator , covered with Parafilm tape.
• The liquid culture tubes were incubated in the shaking incubator at 1415h.
• The incubation conditions: 37˚C, horizontal shaking at 180rpm.

18/07/2015

• The liquid culture inoculated on 17/07 showed good growth based on optical density (visual inspection). Sediment observed in each tube in addition to turbidity in liquid media, indicative of high bacterial concentration.
• Liquid culture tubes were removed from shaking incubator, sealed with Parafilm and placed in 4˚C refrigerator at 1335h.
• Agar plates from transformations carried out on competent cells from Kenan remain in the incubator. Both promoter plates show moderate growth while terminator plates remain clear with no visible growth.

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.

Week 3

26/07/2015

• The 3 transformation tubes containing Promoter2 + Ampicillin, Promoter3+Ampicillin, Promoter1+ Ampicillin, are still clean so we proceeded to discard them.
• After centrifuging the tubes, all the Terminator in Ampicillin tubes have white pellets except Ter2 tube (diluted), which had a red pellet. Hence we discarded Ter2 tube.
• We performed the Miniprep on the other 5 tubes with white pellets.
• Then performed digestion and ligation on the plasmids using standard protocols.
• Then we transformed the DNA into competent cells

Ligation

• Exception for the ligation:
  1. We added 5ul of each digest and ccdb backbone
  2. 2ul of DNA ligase
  3. 4ul of buffer solution but no water
• This was spun down for 10 seconds in the centrifuge.

Transformation:

• The transformation was done according to standard protocol
• However, for step 7, tube 1& 3 are placed in the normal incubator at 37˚C and at 130rpm
• However, for step 7, tube 1& 3 are placed in the normal incubator at 37˚C and at 130rpm
• We created 2 duplicates of transformation plate for each tube.

27/07/2015

• We did not use the correct restriction enzymes on the ccdb cells; hence we had to re-do the ligation and digestion of the plasmids. See above for the protocol ( for the 26/07/15)
• We then followed with the transformation of the plasmids by transforming 5ul each of the transformed plasmids in each tube of the competent cell

Transformation:

The transformation was followed according to standard protocol, with the exception of certain steps:

1. Heat shock the cells at 42˚C.
2. The cells were placed on ice for 3 instead of 5 minutes
3. Pipette 900ul of S.O.C. mixture
4. The tubes were shaken in the heating block at 37˚C, at 250rpm.

28/07/2015

For the stock concentration of antibiotic:

• we need 50ug/mL of Chloroamphenciol ( from earlier documentation)
• target conc: 50uh/mL
  target conc: 50uh/mL
• So we have 300ml of LB and our final conc. needs to be 50ug/mL. Hence we measure out 6mg of antibiotic into the broth.
• So We added 330ul of antibiotic ( of stock concentration 100mg/mL) into 300ml of LB Broth, then pipetted 10ml into each test tube.
• Picked colonies from each plate, inoculated them and left them in the incubator at 250rpm.

29/07/2015

• Miniprepped the plasmids but we accidentally threw out 2 test tubes of DNA but we still had 4 duplicates left.
• We then performed a Nanodrop of the ChiA constructs

Results Of 2nd Nano Drop

Tube	Nucleic acid	Unit	A260	A280	260/280	260/320	Factor
ChiA 3.1	95.2	ng/ul	1.904	0.987	1.93	2.27	50
ChiA 2.1	37.0	ng/ul	0.741	0.379	1.96	1.82	50
ChiA 2.2	360.0	ng/ul	7.200	3.811	1.89	2.26	50
ChiA 3.2	156.3	ng/ul	3.127	1.646	2.02	1.91	50

26/08/2015

• Performed ligation of the promoter and tnaA
• Amplifying the tnaA and tnaB sequence.
• Added 200ul of water (DNA- RNA free) into each tube
• Performed the Nano Drop . The concentration of both tubes is approximately 7-8ug/ul.

27/08/2015

• Nanodrop to verify concentration of resuspended DNA
• Nuclease- free H2O used to resuspend the DNA was found to be contaminated with 4.8ug/ul of DNA
• The actual DNA content of plasmids are around 6-8 ug/ul on average
• We decided to proceed with the transformation since protocol specified 10ug- 100ug of DNA.

30/08/2015

• We made liquid broth, Kanamycin

Preparing agar plates:

1. Take 35g of agar powder in 1L of solution.
2. Add the powder then autoclave the solution( 1hour)
3. We already have antibiotic stock solution of 100mg/ml

Note: Due to coagulation, we had to put in 400ul of antibiotic( chloroamphenicol) instead of 500ul as originally desired.