Lab Plan

Week 16: August 10-16

1. Making of competent cells
2. Colony PCR from clones Flic_MCS BBaJ23_110(4)
3. Agarose Gel of Colony PCR
4. Done Precultures of positive clones
5. Sequencing of positive clones
6. PCR amplification of flic_MCS PCR product 2 (Purification via Gel-Ex)
7. Another Sequencing of positive clones ( missing part of Flic at C-terminus)
8. PRC amplification of flic_MCS vie gradient PCR (all gradient PCR 62-68°C) (used different suffix Primer, since resitriction enzyme has no overhanging ends for digestion)
9. DNA extraction of gradient PCR 62-68°C
10. Digestion of Flic_MCS Gradient 62-68°C
11. Gel EX of digested Flic_MCS Gradient 62-68°C
12. Sequencing of Flic_MCS Gradient 62-68°C( looked good)

Week 17: August 17-23

1. Digestion of backbone BBaJ23_106(1), BBaJ23_110(4), BBaJ23_118(1)
2. Gel-Ex of digested backbones
3. Ligation of digested backbones with digested Flic_MCS Gradient 62-68°C
4. Transformation in chemical competent cells
5. Colony PCR of cells
6. Miniprep of precultures (Determination of concentration)
7. Sent to sequencing
8. Making of delta Filc MG 1655 competent cells
9. Ligation of Flic_MCS with D3 Domains
10. Transformation into delta flic MG 1655 cells (Transformation did not work)

Week 18: August 24-30

1. Ligation of Flic_MCS in PSB1A3, Flic_MCS in PSB1C3, Flic_MCS_106and Flic_MCS_110 in PSB1C3, gB3 in PSB1C3 into delta_Flic MG 1655
2. Motility essay of Flic_106_1_clone1, Flic_110_1_clone13 MG 1655 wildtype
3. Miniprep of precultures
4. Making of electrocompetent DH10B cells
5. Transformation of BioBricks in PSB1C3 and into Flic_MCS_106 and Flic_MCS_110
6. Colony PCR of clones
7. First BioBricks: gB4_110_Flic_MCS_PSB1A3!!!
8. Transformation of clones 1 and 13

Week 19: August-September 31-6

1. Digestion ,Ligation and Transformation of gBlocks into clone 1 and clone 13
2. Miniprep of clones
3. Transformation of gB3-gB7 into Flic_MCS and PSB1C3( did not work, PSB1C3 was not digested properly)
4. Another digestion of PSB1C3 with Gel-EX (EcoRI and PstI)
5. Redone Transformation (Point 3)

Week 20: September 7-13

1. Colony PCR of Transformation ( Took wrong primers 794 instead of 16 for PSB1C3)
2. Miniprep of clones
3. Repeated Colony PCR of Transformation
4. Transformation of gBlocks into clone L2 (clone 13 inPSB1C3)and clone K2 (clone 1 in PSB1C3)
5. Colony PCR
6. Motiliy Essay
7. Transformation of M2(clone 14.2 (gB3) into PSB1C3) into delta Flic in MG 1655, wildtype and B834

Week 21: September 14-20

1. Miniprep of precultures
2. Sent to sequencing
3. Digestion, Ligation and Transformation of gB4,5 and 6 into clone 1 and clone 13
4. Miniprep of clones
5. Colony PCR of clones ( BioBricks gB5 and gB6 into Flic_MCS PSB1C3 looks good)
6. Isolation of Flagellas mechanically and chemically
7. SDS-Gel of isolation ( did not work, no flagellas via chemical isolation, cell lysis via mechanically treatment)

Cellulose Lab Plan

13.5.15
● Prepare 1L of liquid (bottle) and 250ml of solid medium (plates)
18.5.15
● Streak 6 plates:
○ 2x liquid kombucha
○ 2x pellicle from kombucha
○ 2x negative control plates
● Incubate at 26C for 3 days
● Result: Plates were dehydrated. Repeat isolation
27.5.15
● Repeated plates were positive. Single fresh looking colonies were streaked again in solid plates for storage and also in liquid medium for pre-cultures
1.6.15
● Innoculation of pre-cultures into different liquid volumes including Erlen Meyers, Falcon tubes and sectioned petri dishes.
● 400ml with 1-5ml of liquid pre-culture
● 250ml with 1ml of pre-culture
● 15ml with pellicle
● 2X ¼ of petri dish with pellicle
● 1X ¼ of petri dish with 1ml of culture
● 2x petri petri dish with 1ml of culture
● 2x petri dish with pellicle
● Incubate at 26C for 5-7 days
3.6.15
● All liquid cultures were positive and presented cellulose on the surface.
● The cellulose has taken most of the liquid medium in the petri dishes
● The 400ml and 150ml showed growth but the cellulose looked like needed more time to thick more and grow more.
● 2 L of medium prepared
4.6.15
● Analysis of liquid cultures: cultures inoculated with liquid pre-cultures showed a homogeneous growth meanwhile cultures inoculated with pellicle showed the site of inoculation and did not look very homogeneous.
● Some cultures from the sectioned petri dishes (triangle shape) were dried between glasses for the Long Night of Sciences exhibition.
8.6.15
● New inoculations in 50ml of medium in a 250ml Flask.
● 1ml, 500ml, 100ml, 10ml, pellicle of pre-culture
● Cellulose from 400ml and 150ml flasks started on 1.6.15 were washed in distilled water for 48 hours.
9.6.15
● Create a new medium stock but filtering the cellulose separately and adding it to the autoclaved solution afterwards.
● Innoculate in 50ml of medium in a 250ml Flask:
○ 1ml, 500ml, 100ml, 10ml, pellicle of pre-cultures
● Wash cellulose for previous cultures in NaOH for 24 hours.
10.6.15
● Dry previously washed cellulose with towel paper and manually by putting pressure on it and squeezing it.
● Harvest and wash cultures that look ready inoculated previously.
● Inoculate a 500ml flask filled with 100ml of media with 2ml of pre-culture.
11.6.15
● New big cultures started (same setting as last day)
15.6.15
● Cultures harvested and washed with water and later with 0.1% NaOH
● 40x liquid cultures (on petri dishes) started from pellicle and incubated at 30°C
● 3x new pre-cultures started in 5ml (falcon)
17.6.15
● Ready cellulose from previous cultures left overnight in water or 0.1%NaOH
● 40x liquid cultures in petri dishes did not look ready for harvesting
18.6.15
● New 2 pre-cultures from soft fresh looking colony P0 and hard colony P0. Incubate at 30°C
● Big open surface cilinder inoculated with 5-10ml of pre-cultures and incubated at 30C.
● 40x cultures harvested, washed and left overnight in 0.1% NaOH. They were later stored in water at 4°C
25.6.15
● Ready cultures left overnight in 0.1% NaOH
● 40x pelets stored in water, washed again
● Big culture from cillinder (aprox 20cm diameter, 0.5cm tall) left overnight in 0.1% NaOH after washing.
29.6.15
Manual dry of part of cellulose available and further dehydrated at 30°C
1.7.15
● More cellulose dryed manually. Half of it left at 80C. Other half at room temperature (this one eventually did not get dry so some of it was stored wet and the remaining was dried at 80C)
● Production of 4L of medium with glucose filtered separately.
2.7.15
● Check cellulose dryed at 80°C ● Room drying cellulose looked like it needed more time
● Inoculate 2 liter of medium aliquoted in several tupperwares. Tried to achieve the biggest surface with not so much volume.
● 10g of dried cellulose obtained after drying. Stored at 4°C
6.7.15
● 10g of cellulose initially blended with 60ml of water with manual blender
○ The procedure lasted up to 1 hour with constant manual homogenisation of the mix.
● Blender overheated
● Another 10g of cellulose left at room temperature needed to go into the oven.
18.7.15
● New cultures started from agar cultures P0
● Ready cultures were harvested, washed and left overnight with 0.1% NaOH
● Cellulose was autoclaved with distilled water
● Pulverised cellulose was left on petri dished to see if it can coat it. It coated them uniformingly and even. A very thin layer was observed.
● Not pulverised cellulose was dried at 80C and then re-introduced in water to see if it will rehydrate. It rehydrated just a little bit. Not significant.
● Water was added to coated petri dishes to see if the coating will resuspend. It did only a little bit after scratching.
6.8.15
● Introduction to use the fluorescent plate reader
10.8.15
● Coating and experiment with different concentrations of cellulose from bacteria, paper and powder.
● Plate dried at 37°C
18.8.15 and 19.8.15
● Dilution of CBD-GFP in buffer to see if plate reader can detect it. It was detected and the optimal dilution was 1:10
● Binding took place
25.8.15
● Coat wells in triplicates and drying
27.8.15
● Match the CBD with the CBD-GFP concentrations by dilution. The concentration achieved was 0.5XX for both at 490nm.
31.8.15
● Measure fluorescense only with PBS.
● Bind overnight on paper, powder and bacterial cellulose a 4C on triplicates
1.9.15
● Read plate with solution, without solution, with PBS y subsequently after every wash.
● Coat wells only with paper and bacterial cellulose.
2.9.15
● Perform dilutions for binding and perform reading.
● Bind overnight
● Results in Excel