Lab Plan

Week 1: April 27- May 1st

 preparations for our experiments
- solutions for making competent cells, LB medium, LB agar plates w/wo AB
- AB stocks
 streakouts and preculturesand preculture of strains DH10b, B834, MG1655, BL21gold
 MG1655 and BL21 gold prepared as chemically competent cells
 DH10b and B834 prepared as electro competent cells

Week 2: May 04-08

 Transformation of Biobricks, (listed below) and PSB1C3 into electro competent cells of strain DH10b
 BBa_K936013 = pelB_cutinase fusion protein
 BBa_K1321348 = sfGFP fused to CBD
 Isolation and sequencing of all constructs

Week 3: May 11-15

 Digestion and cloning of:
1) pSB1C3 (backbone) + sfGFP_dCBD (insert)
2) pSEVA
with EcoRI and SpeI. change of strategy because insilico check revealed missing ribosomal binding site.
 New strategy: cloning of sfGFP_dCBD into pEX_HisII instead of pSEVA
 Transformation and isolation of pEX_HisII
 Digestion of pEX_HisII with AgeI and PstI
 Digestion of sfGFP_dCBD_PSB1C3 with NgoMIV and PstI
 Gel-Extraction for purification of insert sfGFP_dCBD and backbone pEX_HisII
 ligation o/n and transformation into DH10b and BL21gold

Week 4: May 18-22

 Screen for green fluorescent clones
- colony PCR with analytical gel
 Precultures and Miniprep of promissing clones

Week 5: May 25-31

 Precultures of:
 MG1655 Z1
 MG1655 Z1 ΔfliC
 DS941 Z1
 DS941 Z1 ΔfliC
 Precultures of positive clones from colony PCR (pEX_HisII_sfGFP_dCBD)
 Transformation of pEX_HisII_sfGFP_dCBD into BL21gold
 Precultures for test expression of one clone from colony PCR (pEX_HisII_sfGFP_dCBD)

 Kryo stocks of:
 3x MG1655 Z1
 3x MG1655 Z1 ΔFliC
 3x DS941 Z1
 3x DS941 Z1 ΔFliC
 Clone 1, 2, and 3 (ColonyPCR)
 Started test expression with 6 different conditions:
a. 37°C, induction with 0.5 mM IPTG
b. 37°C, induction with 1 mM IPTG → best condition
c. 30°C, induction with 0.5 mM IPTG
d. 30°C, induction with 1 mM IPTG
e. 26°C, induction with 0.5 mM IPTG
f. 26°C, induction with 1 mM IPTG
 SDS-PAGE analysis of samples from test expression

Week 6: June 01-05

 large scale expression sfGFP_dCBD (induction with 1 mM IPTG o/n)
 harvesting and cell lysis
 protein purification by Ni-NTA chromatography
 purification process was stopped → His-tag seemed not to bind to the column)

Week 7: June 08-12

 Preculuture of BL21gold transformed with pEX_HisII_sfGFP_dCBD (for repeating the large scale and purification process)
 large scale expression sfGFP_dCBD (induction with 1 mM IPTG o/n)
 harvesting, cell lysis (Microfluidizer) and protein purification by ÄKTA system purification failed (GFP was collected in the flow-through)
 SDS-PAGE analysis

Week 8: June 15-19

 Precultures of successful pEX_HisII_sfGFP_CBD clones from patching plate of colony PCR
 Cloning not successful

Week 9: June 22-26

 PCR of BBa_K1321348 (sfGFP_dCBD)
 Purification of PCR product
 Gibson Assembly of ordered gBLOCKS (FliC_MCS) (NEBuilder HiFi DNA Assembly Reaction Protocol) + analytic agarose-gel
 Cloning of sfGFP_dCBD into pEX_HisII repeated
 Gel-ex for purification
 Chemical transformation into BL21gold

Week 10: June 29 – July 3

 Gibson Assembly Reaction of FliC_MCS repeated
 PCR amplification of FliC_MCS
 Sequencing of FliC_MCS
 Colony PCR of ligated sfGFP_dCBD and pEX_HisII
 Gel-Ex of colony PCR products
 Miniprep + sequencing
 Transformation of pEX_HisII plasmid into BL21gold cells + miniprep
 Restriction digestion of FliC_MCS, PSB1C3, 2x sfGFP_CBD
 PCR purification of FliC_MCS and PSB1C3
 Ligation of FliC_MCS with pEX and FliC_MCS with PSB1C3 (mistake found → wrong restriction enzymes were used)

Week 11: July 06-10

 Precultures, miniprep, sequencing of pEX_HisII_sfGFP_dCBD
 PCR amplification of 5 gBLOCKS (gb 3-7) coding for different D3 domains
 Gibson assembly of FliC_MCS repeated again
 PCR purification of gb3-7 and FliC_MCS
 Chemical transformation of Biobricks (promoters) BBa_J23106, BBa_J23110 and BBa_J23118 into Bl21 gold, miniprep, sequencing
 Digestion of FliC_MCS XbaI and PstI
 Precultures pEX_HisII_sfGFP_dCBD for large scale expression

Week 12: July 13-17

 Preparation of buffers for protein purification
 Large scale expression (1mM IPTG induction)
 Harvesting and Lysis of cells
 Purification with His Trap FF (fast flow)
 SDS gel of large scale expression + purification samples
 Digestion of Biobricks (EcoRI+SpeI), FliC_MCS (XbaI+PstI), PSB1C3 (EcoRI+SpeI)
 Analytic gel (digestion not successful because RFP not cutted out)  repeated with EcoRI+PstI (PstI instead of SpeI) and Gel-ex
 Ligation of FliC_MCS with BBas (J23106, J23110, J23118)
 Chemical transformation of ligation mixes into Bl21 gold
 Precultures + miniprep + sequencing

Week 13: July 20-24

 Made DH10b electro competent cells
 Digestion of FliC_MCS + purification
 Ligation of BBa_J23106 and BBa_J23110 with FliC_MCS
 Transformation into DH10b

Week 14: July 27-31

 Colony PCR of trafos + analytic gel + precultures + miniprep + sequencing
 Transformation into DH10b
 Repeated week 13 because of bad sequencing results

Week 15: August 03-07

 Repeated week 13/14 because still mismatches in sequencing results
 Changed strategy of PCR amplification to gradient PCR

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