iGEM 2009 — Team Lethbridge

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# Team:Lethbridge/Notebook

### From 2009.igem.org

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* [Home](https://2009.igem.org/Team%3ALethbridge)
  + [Project Overview](https://2009.igem.org/Team%3ALethbridge#Our_Project:_The_BioBattery)
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* [Team](https://2009.igem.org/Team%3ALethbridge/Team)
* [Project](https://2009.igem.org/Team%3ALethbridge/Project)
  + [Project Details](https://2009.igem.org/Team%3ALethbridge/Project#Project_Details)
  + [Experiments](https://2009.igem.org/Team%3ALethbridge/Project#The_Experiments)
  + [Results](https://2009.igem.org/Team%3ALethbridge/Project#Results)
* [Ethics](https://2009.igem.org/Team%3ALethbridge/Ethics)
  + [Ethics and Synbio](https://2009.igem.org/Team%3ALethbridge/Ethics#Ethics_and_SynBio)
  + [The Survey](https://2009.igem.org/Team%3ALethbridge/Ethics#The_Survey)
* [Notebook](https://2009.igem.org/Team%3ALethbridge/Notebook)
  + [Calendar](https://2009.igem.org/Team%3ALethbridge/Notebook#Calendar)
  + [May](https://2009.igem.org/Team%3ALethbridge/Notebook#May)
  + [June](https://2009.igem.org/Team%3ALethbridge/Notebook#June)
  + [July](https://2009.igem.org/Team%3ALethbridge/Notebook#July)
  + [August](https://2009.igem.org/Team%3ALethbridge/Notebook#August)
  + [September](https://2009.igem.org/Team%3ALethbridge/Notebook#September)
  + [October](https://2009.igem.org/Team%3ALethbridge/Notebook#October)
* [Meetings](https://2009.igem.org/Team%3ALethbridge/Team_Meetings)
  + [April](https://2009.igem.org/Team%3ALethbridge/Team_Meetings#April)
  + [May](https://2009.igem.org/Team%3ALethbridge/Team_Meetings#May)
  + [June](https://2009.igem.org/Team%3ALethbridge/Team_Meetings#June)
  + [July](https://2009.igem.org/Team%3ALethbridge/Team_Meetings#July)
  + [August](https://2009.igem.org/Team%3ALethbridge/Team_Meetings#August)
  + [September](https://2009.igem.org/Team%3ALethbridge/Team_Meetings#September)
  + [October](https://2009.igem.org/Team%3ALethbridge/Team_Meetings#October)
* [Modeling](https://2009.igem.org/Team%3ALethbridge/Modeling)
* [Parts](https://2009.igem.org/Team%3ALethbridge/Parts)
  + [Favourites](https://2009.igem.org/Team%3ALethbridge/Parts#Favourites)
  + [Individual Parts](https://2009.igem.org/Team%3ALethbridge/Parts#Individual_Parts)
  + [Constructs](https://2009.igem.org/Team%3ALethbridge/Parts#Constructs.2FDevices)
  + [Intermediates](https://2009.igem.org/Team%3ALethbridge/Parts#Intermediate_Parts)
* [Collaboration](https://2009.igem.org/Team%3ALethbridge/Collaboration)
* [Judging](https://2009.igem.org/Team%3ALethbridge/Criteria)
* [Extra Information on SynBio](https://2009.igem.org/Team%3ALethbridge/videos)

## Calendar

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

## May

### May 5

**Roxanne, Megan, Alix, Mackenzie, Sebastian**

* prepared Lb Media in test tubes -prepared 250mL LB agar for plates

* Prepared 500nM Tris-HCl (pH 8.0), EDTA (pH 8.0, 500nm) TE Buffer (pH 8.0), GTE (Solution I), 2M NaOH, 10% SDS

### May 6

**Roxanne**

* Autoclaved LB test tubes, LB agar, Tris-HCl, EDTA, TE, GTE

* Added ampicillin to LB agar (100ug/mL) -Poured LB + amp plates

### May 12

**Roxanne**

* Prepared LB agar
* added Tetracyclin (100ug/mL) –
* Poured LB + ampt plates

Roxanne, Alix, Ashley, Megan, Mackenzie, Kirsten

* transferred pE43 (DH5α) cells from gylcerol stock to 5mL LB+Tet test tube (x8)
* placed in orbital shaker overnight at 37°C

### May 13

**Roxanne**

* Removed test tubes from shaker (16hrs incubation) and placed in deli fridge

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### May 14

**Roxanne, Alix, Ashley, Mackenzie, Kirsten**

* Preformed alkaline lysis miniprep on pE43 plasmid -lost plasmid from 2 tubes

* obtained 6X pE43 plasmid (2X ohbR, 2x ohbB, ohbA, ohbC)

### May 19

**Roxanne, Alix, Ashley, Kirsten, Megan, Mackenzie**

* Gel Electrophoresis : 1% agarose in TAE

Lane 1: Ladder

Lane 2: ohbR1

Lane 3: ohbR2

Lane 4: ohbC

Lane 5: ohbB1

Lane 6: ohbB2

Lane 7:ohbA

Lane 8:ohbC

Lane 9: ohbB1

Lane 10:ohbB2

* DNA quantification by UV spec 260nm : 2.013 280nm: 1.105 260/280 : 1.984 Concentration: 2517µg/mL PCR of ohbA, (ohbB)x2, ohbC, (ohbR)x2

* Took picture of the gel - it did not turn out.

### May 21

**Roxanne**

* Made 50X TAE Buffer
* Running PCR amplicons & RS/pSB1A2 ligation product on a 1% agarose gel
* Jeff gave tutorial on primer design and quick-change mutagenesis to Kirsten, Ashley, Mackenzie, Fan, Lisza, Roxanne

[![Ohb PCR-Phusion (Rox).JPG](/wiki/images/6/6c/Ohb_PCR-Phusion_%28Rox%29.JPG)](/File%3AOhb_PCR-Phusion_%28Rox%29.JPG)

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### May 26

**Roxanne, Alix, Megan, Mackenzie, Kirsten**

* Gel did not turn out gave tutorial on open wetware and the igem registry

### May 28

**Roxanne**

* reran the gel from may 19th, only primer dimers present

[![Ohb PCR (Roxanne).JPG](/wiki/images/d/de/Ohb_PCR_%28Roxanne%29.JPG)](/File%3AOhb_PCR_%28Roxanne%29.JPG)
**Roxanne, Ashley, Alix, Kirsten, Mackenzie, Megan**

* Redoing the PCR of ohbA, ohbB, ohbC and OhbR with Phusion DNA Polymerase

**Roxanne**

* refilled the tip boxes

### May 29

**Roxanne**

* prepared glycerol stocks for autoclaving

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

## June

### June 1

**Roxanne**

* Ran a 1% agarose in TAE gel of the PCR products from May 28th.
* 5µL of ladder, 2µL of DNA

Lane 1: ladder

Lane 2: ohbA (conc.1x)

Lane 3: ohbA (conc.1/10)

Lane 4: ohbB(1x)

Lane 5: ohbB(1/10)

Lane 6: ohbC(1x)

Lane 7: ohbC(1/10)

Lane 8: ohbR (1x)

Lane 9: ohbR(1/10)

Lane 10: ohbR(1x)

* Gel did not show any amplification

Mackenzie & Roxanne

* Transformation of pSB1A3 into DH5α

* Made 2 aliquots of pSB1A3 and 1 of pUC19

* plated pSB1A3-1: straight and dilute, pSB1A3-2: straight and dilute, pUC19: straight only incubated overnight at 37°C

### June 3

**Roxanne**

* positive control worked
* Think the transformation worked but slight problem. The plasmid contains a suicide gene. Therefore, any cells that took up the plasmid would die.
* planning new genes to transform
* GENE LOCATION pSB1A3 + IPTG Inducible RFP 1-1k Bba\_I13522, pTET GFP 2-8A Bba\_I13521, pTet mRFP 2-6O Bba\_I13600, pTET CFP 1-24E BBA-B0015, double terminator 1-23L

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### June 4

* Picked some glycerol stock GFP for the AIF visit

* made ampicillin plates 371.39g/mol X100mmol/LX0.002L = 74.278mg

### June 15

Transformed the pTet and EYFP genes from the Biobrick registry

### June 16

* Picked colonies in the early morning for Mini prep in the afternoon

* Restriction digested the plasmids: pTet with SpeI and PstI, EYFP with XbaI and PstI

### June 17

* Quenched the restriction digests from the day before

### June 18

* Checked the concentrations of:
  + Riboswitch-1 : 4549u/.mL
  + Riboswitch-2 : 4283ug/mL
  + rpsA TIR-1 : 106ug/mL
  + rpsA TIR-2 : 90ug/mL
  + rpsA TIR-3 : 78ug/mL
  + rpsA TIR-4 : 74ug/mL
  + rpsA TIR-4b : 67ug/mL
  + rpsA TIR-5 : 70ug/mL

in order to send for sequencing with the UR and UF2 primers

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### June 22

* Picked one colony each from DH5a + pTet and DH5a + EYFP.
* Grown overnight at 37 degrees in LB +100ug/mL Amp (5mL culture tubes)
* From mini-preps of EYFP(1&2) and pTet (1&2) did preparative restriction digests
  + pTet with PstI and SpeI
  + EYFP with XbaI and PstI
* In 37 degree water bath for 1hr and 20 min. Quenched at 60 degrees for 15 minutes.

### June 23

* Ran a 1% agarose gel (1mL 50X TAE with 49mL Milli-Q H2O and 0.5g of agarose).

Lane 1: 1kb ladder

Lane 2:EYFP1A

Lane 3: EYFP1B

Lane 4: EYFP1B runover

Lane 5: EYFP2A

Lane 6: EYFP2B

Lane 7: pTet-1A

Lane 8: pTet-1B

Lane 9: pTet2-A

Lane 10: pTet-2B

* Samples were 2uL of dye and 10uL of DNA.
* The gel was unsuccessful. Possible reasons include: too short of an incubation during restriction digest or the enzymes were not functioning properly

[![June 24 (June 23rd work).JPG](/wiki/images/2/29/June_24_%28June_23rd_work%29.JPG)](/File%3AJune_24_%28June_23rd_work%29.JPG)

### June 24

* Mini-Preps of pTet and EYFP
  + 750uL of culture from the fridge into microcentrifuge tubes. Pellet at 13000rpm for 2 min. Removed supernatant and added the rest of the culture. Repelleted at 13000rpm for 2 min.
  + followed protocol according to Qiagen mini-prep protocol on page 2
* Restriction Digest of EYFP and pTet
  + EYFP restricted with XbaI and PstI
  + pTet restricted with PstI and SpeI
    - Double digest:1uL buffer tango, 8uL DNA, 0.5uL of each enzyme
    - Single digest (1 for each enzyme): 0.5uL water, 1uLbuffer tango, 8uL DNA, 0.5uL enzyme
    - Negative control: 1uL water, 1uL buffer, 8uL DNA
    - 8 tubes total in to the thermocycler for 8hrs at 37, 15min at 60 then held at 4 before going into the -20 fridge.

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### June 25

* Ran a gel of the digested EYFP and pTet and controls from the previous day.
* Used 0.3g of agarose in 30mL of 1X TAE
* made stock solution of 1x TAE
* Ran gel at 100V for 1hr

Lane 1: 1kb ladder

Lane 2: Control-no enzyme(pTet)

Lane 3: pTet-pstI

Lane 4: pTet-SpeI

Lane 5: pTet-SpeI/PstI

Lane 6:EYFP-no enzyme

Lane 7: EYFP-PstI

Lane 8: EYFP-XbaI

Lane 9: EYFP-XbaI/PstI

Lane 10: 1kb ladder

* Gel melted
* Made 2 pTet and 2EYFP 5mL culture tubes with LB and Amp(100mg/mL). Left in incubator overnight at 37°C

### June 26

* Cells harvested from pTet and EYFP cultures into 4 tubes. Spun down and LB supernatant removed. Stored in -20 for future use.

### June 30

* 200mL cultures of cells with pTet, EYFP and CFP grown in LB and 100ug/mL Amp harvested by centrifugation then maxi-prepped.
* Maxipreps
  + incubated cell pellets in 3mL of ALSI and incubated at RT for 15min
  + 6mL of ASL2 added and incubated at RT for 10min
  + 4.5mL of AlS3 added and placed on ice for 10min
  + Spun at 5000g at 4 for 15min
  + 5mL of each phenol and chloroform added to tubes containing resulting supernatant
  + spun for 10min at 4 and 5000g, upper aqueous layer saved
  + 5mL of chloroform added to aqueous layer and spun for 10 min at 4 and 5000g. Supernatant saved
  + 0.6 volumes isopropanol added to each tube and placed in -20 freezer for 30min –
  + spun for 10min at 4 and 5000g
  + pellet wasted with EtOH and airdried overnight

* 5 culture (500mL) flasks of LB made: 10g tryptone, 5g yeast extract, 10g salt and 1L water

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

## July

### July 2

* DNA samples dissolved in 1000uL water, 100uL of RNAse added. Made 1/10, 1/100 and 1/1000 dilutions of pTet, CFP and EYFP.

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### July 3

* Ran a 1% agarose gel at 120V for 60 minutes.
* 2uL DNA samples with 2uL 6x loading dye

Lane 2: 10uL of 2kb ladder,

Lane 4:undiluted pTet

Lane 5: undiluted CFP

Lane 6:undiluted EYFP

Lane 8: 1/10pTet

Lane 9: 1/10 CFP

Lane 10: 1/10 EYFP

Lane 12: 1/100 pTet

Lane 13: 1/100 CFP

Lane 14: 1/100 EYFP

Lane 16: 1/1000pTet

Lane 17: 1/1000 CFP

Lane 18: 1/1000 EYFP

[![July 3.png](/wiki/images/1/12/July_3.png)](/File%3AJuly_3.png)

### July 6th

**Jeff, Mackenzie, Ashley**

Using 1/10 and 1/100 dilutions of pTET and EYFP to perform large scale digests.

* pTET:PstI and SpeI
* EYFP:XbaI and PstI

4 reactions each:

* single digest (PstI/SpeI and XbaI/PstI)
* double digest
* no digest

100µL each, means we need 400µL total for each.

1/10 dilution=40µL DNA, 360µL water

1/100 dilution=4µL DNA, 396µL water

Reaction set up:

* 88µL DNA
* 10µL buffer Tango (10x)
* 1µL RE1 (or water)
* 1µL RE2 (or water)
* =100 µL total

16 reactions run overnight @ 37° C

**Kirsten, Lisza**

TetR Q04400-plate 1 well 16p-pSB2K3

LacI promoter-R0010-plate 1 well 1d-Amp

pBAD-I13453-plate 1, well 1n-pSB1A2

strong RBS-B0030-plate 1 well 1h-pSB1A2

med RBS-B0030-plate 1 well 2I-pSB1A2

10µL water into wells

Transformation:

2µL DNA (TetR, LacI, pBAD, med RBS, strong RBS) into 25µL competent DH5α cells (from the -80 fridge).
Pipet up then down and swirl. One ice for 30min, then heat shock at 42° C for 45 sec. Ice for 1 min, add 250µL LB broth (from fridge) and incubate in shaker for 1 hour. Plate onto ampicillin plates (pBAD, LacI, strond and med RBS) and kanamycin plate (TetR) into incubator overnight. Control: 2.5µL water and 2.5µL DH5α.

### July 8

**Jeff**

Overnight cultures (200mL + antibiotics) of:

* PlacI (Amp)
* TetR (Kan)
* Med RBS (Amp)

Centrifuged at 5000g, 10 min, 4° for maxiprep

500µL each of each culture used to make glycerol stocks – duplicates made, stored in -80° freezer after flash freezing with liquid nitrogen.

EYFP insert from AGE separation (incubated in 600µL buffer QG overnight):

* +200µL isopropanol
* Incubated on a QiaQuick column
* 13000rpm, 1 min

Flow through replaced in original tube. 500µL QG applied to column, 1 min centrifugation. Colum washed w/ 750µL PE buffer (1 min centrifuge). Flow through discarded. 1 more min of centrifugation to get rid of buffer PE. DNA eluted w/ 25µL water (37°C for 10 min) also a second water elution w/ 25µL water (E2).

1/10 double digest of pTet heat incubated at 85°C for 10 min.

Maxipreps: 3mL ALSI added, cells responded. 500µL lysozyme added. 15 min @RT, 6mL ALSII added. 10 min at RT. 4.5mL cold ALSIII added, 10 min on ice. Left a 4°C

### July 13

Setting up ligations:

1µL ligase (stock)

10x T4 DNA ligase buffer

1/10 double digested pTet est 20ng/µL

E1 (60% yield) = ~5ng/µL; ~1/3 insert size/pTet

Control needed:

* 1/10 double digested pTet, no ligase
* 1/10 double digested pTet + ligase
* 1/10 double digested pTet + 3x vol of insert
* 1/10 double digested pTet +6x vol of insert

Reaction 1:

* 1µL T4 DNA ligase 10x buffer
* 1µL double digest pTet
* 8µL water

Reaction 2:

* 1µL T4 DNA ligase 10x buffer
* 1µL double digest pTet
* 0.5 µL T4 ligase
* 7.5µL water

Reaction 3:

* 1µL T4 DNA ligase 10x buffer
* 1µL double digest pTet
* 0.5 µL T4 ligase
* 3µL E1 EYFP insert
* 4.5µL water

Reaction 4:

* 1µL T4 DNA ligase 10x buffer
* 1µL double digest pTet
* 0.5 µL T4 ligase
* 6 µL E1 EYFP insert
* 1.5µL water

Incubated at 37°C overnight

Kirsten, Mackenzie
Chloroform extraction of medRBS, pLacI, TetR, that Jeff started.
pBab & strong RBS cloudy and phenol chloro done twice.

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### July 14

**Megan, Mackenzie, Alix**

Transformations of ligation reactions

Took 2µL of DNA from 1, 2, 3, 4 and added to 25 µL of DH5α cells. Incubated on ice for 30 min. Heat shocked at 42°C for 45 sec. Back to ice for 1 min. For ligations add 500µL of LB broth to each tube, working sterile using a Bunsen burner. Incubate in shaker at 37°C for1 hour at 200 rpm. Made up two plates (Amp) of each, 100µL and 400µL.

### July 16th

**Ashley, Mackenzie, Roxanne**

Pelleted pTet-EYFP ligation reactions 3 and 4 using at 13000rpm for 1 min.

Mini-prep of pTet-EYFP ligation reactions 3 and 4 using Qiagen kit + protocol.

### July 20th

**Lisza**

Our genes: added miiliQ water

* 10µL to N-term Arg tail, concentration = 200ng/µL
* 10µL to c-term Arg tail, concentration= 200ng/µL
* 20 µL to lumazine, concentration= 250ng/µL

### July 21st

**Lisza**

Made glycerol stocks of the 4 pTet-EYFP ligations

**Kirsten, Alix**

Mini-prep of pTet-EYFP (3 &4)x2 followed protocol according to Qiagen.

Analytic restriction digest of above mini-preps

Control used mini prepped pTet from June 24.

Added (in order) to 1.5mL micro centrifuge tubes:

* 12µL water
* 2 µL buffer tango
* 5 µL DNA
* 0.5µL XbaI
* Total: 20 µL

**Megan, Kirsten, Alix, Lisza**

Made an 80mL agarose gel for large rig

0.8g agarose

80mL 1x TAE

Lanes

1. Ladder 1Kb
2. Blank
3. pTet unrestricted
4. pTet undigested
5. rxn III-1 undigested (pTet-EYFP)
6. rxn III-1 digest
7. rxn III-2 undigested
8. rxn III-2 digest (XbaI)
9. blank
10. rxn IV-1 undigested
11. rxn IV-1 digest (XbaI)
12. rxn IV-2 undigested
13. rxn IV-2 digest (XbaI

Ran at 120V for 40 min( in @ 5:45) stained in ethidium bromide for 10 20 min.

Picture:

[![July21pTetEYFP.jpg](/wiki/images/5/5b/July21pTetEYFP.jpg)](/File%3AJuly21pTetEYFP.jpg)

**Lisza**

Made glycerol stocks of maxiprep cultures:

3 lumazine

2 Arg N-term

1 Arg c-term

Spun down cultures

Lumazine-1=1.35g

Lumzine-2= 1.31g

Lumazine-3=1.29g

Arg c-term=1.26g

Arg n-term-1=1.43g

Arg n-term-2=1.12g

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### July 23

Did a colony screen PCR:

24 colonies from July 14th DH5α Amp

AB+MC+MT plates labeled 10-32

With picked colonies transferred bacteria into a 1.5mL tube with 300µL milliQ water. With same toothpick inoculated 5mL (Amp 5mL) grown overnight at 37°C. Put 1.5L tubes onto heat block at 95°C for 7 min. Centrifuged at full speed for 1 min. Used 5µL supernatant for PCR survey.

Reactions: control with EYFP

* 10.4µL water (14.9 µL control)
* 2 µL Taq buffer
* 0.4 µL dNTPs
* 1 µL VR primer
* 1 µL VFZ primer
* 0.2 µL Taq polymerase
* 5 µL template (0.5 µL control)

Cycle: Colony58

Note: tube 11 contained both samples 11 and 12

### July 24th

1% agarose gel at 100V of colony PCR

Lanes:

1. 1 kb Ladder (8 µL)
2. Control
3. Colony 10 (10 µL sample+2 µL dye)
4. Colony 11/12
5. Colony 13
6. Colony 14
7. Colony 15
8. Colony 16
9. Colony 17
10. Colony 18
11. Colony 19
12. Colony 20
13. Colony 21
14. Colony 22
15. Colony 23
16. Colony 24
17. Colony 25
18. Colony 26
19. Colony 27
20. Colony 28
21. Colony 29
22. Colony 30
23. Colony 31
24. Colony 32
25. Colony 33
26. 1 Kb ladder (8 µL)

[![July 24 colony screen.png](/wiki/images/9/9e/July_24_colony_screen.png)](/File%3AJuly_24_colony_screen.png)

PCR was successful, but had negative colonies. No pTet EYFP biobrick, only pTet was in the plasmid

### July 27

**Fan & Lisza**

Preparative restriction digest of pTet-EYFP:

* 50µL reactions
* 40 µL DNA
* 5 µLbuffer
* 0.5 µL RE1
* 0.5 µL RE@
* 4 µL milliQ water

pTet:

* RE1=pstI
* RE2=speI

EYFP:

* RE1=pstI
* RE2=xbaI

Incubated at 37°C for 2 hours

**Kirsten, Lisza, Mackenzie**

Purified double digested pTet using QiaQuick PCR purification kit. First run through eluted DNA with 50 µL water then done again and eluted with 30 µL elution buffer. Into -20°C fridge

Gel Extraction of EYFP

Weight of tube: 1.0973g

Weight of tube+gel: 1.9759g

Weight gel: 0.8786

Transferred to falcon tube

Preformed gel extraction according to Qiaquick Gel Extraction kit. Eluted with 30 µL of elution buffer.

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### July 28

**Kirsten, Alix, Lisza**

Ligation of pTet and EYFP

Reaction 1

* 1.55µL of water
* 2.5 µL 10x buffer
* 5 µL vector (pTet)
* 14.7 µL insert (EYFP)
* 1.25 µLT4 DNA ligase
* 25 µL total

Reaction 2 (Control)

* 16.25µL of water
* 2.5 µL 10x buffer
* 5 µL vector (pTet)
* 0.0µL insert (EYFP)
* 1.25 µLT4 DNA ligase
* 25 µL total

Incubate at room temperature for 2 hours then transform

Restriction Digest of Lumazine 1&2, C-terminal Arg tag, N-terminal Arg tag 1&2.

* 14.6µL of water
* 2.5 µL 10x buffer tango
* 4 µL DNA
* 0.4µ EcoRI
* 21 µL total

Incubate at 37°C for 2 hours in water bath.

### July 28

**Fan**

Did a transformation with ligation reactions plus a water control.

### July 29

Running gel of the PCR for EYFP and CFP with c-terminal fusion tags and the restriction digests of n-terminal, c-terminal ARG tags and the lumazine gene.

Loading 6µL ladder, 5µL sample+1µL dye

Lane:

1. 1kb DNA ladder
2. CFP amplicon
3. EYFP amplicon
4. Empty
5. N-terminal #1 (EcoRI digestion)
6. N-terminal #2 (EcoRI digestion)
7. C-terminal (EcoRI digestion)
8. Lumazine # 1 (EcoRI digestion)
9. Lumazine # 2 (EcoRI digestion)

[![July29.png](/wiki/images/a/a5/July29.png)](/File%3AJuly29.png)

Did colony PCR of the 5 colonies from the EYFP-1 plater, 5 of the EYFP-2 plate, 4 of the pTet-1, 4 of the pTet-2, one negative control and one positive (EYFP plasmid). Followed protocol of July 23.

**Megan, Lisza, McKenzie**

Purify maxipreps using kit. Finished 60 µL of PCR samples with a 5 buffer to 1 PCR sample ratio. Buffer amount = 300 µL. Followed protocol in kit.

Ran a gel with ladder, 3 wells for PCR, 3 double wells for extraction

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### July 30

**Lisza**

PCR for fusion c-terminal suffix

Control: no template DNA

* 1.4 µL milliQ water
* 4 µL buffer (5x Phusion)
* 0.4 µL 10mM dNTPs
* 1 µL sense
* 1 µL antisense
* 0 µL DNA
* 0.2 µL phusion polymerase

One for CFP and one for EYFP:

* 11.4µL milliQ water
* 4 µL buffer (5x Phusion)
* 0.4 µL 10mM dNTPs
* 1 µL sense (prefix primer)
* 1 µL antisense (antisense FP Fusion c-term tag)
* 2 µL DNA
* 0.2 µL Phusion polymerase

One for CFP and one for GFP:

* 28.5µL milliQ water
* 10 µL buffer (5x Phusion)
* 1 µL 10mM dNTPs
* 2.5 µL sense (prefix primer)
* 2.5 µL antisense (antisense FP Fusion c-term tag)
* 2 µL DNA
* 0.5 µL Phusion polymerase

Control: no polymerase (water= 29µL)

**Roxanne**

1% agarose gel in TAE of the colony PCR

E1= plate 1 of EYFP ligation

E2= plate 2 of EYFP ligation

(-)= no template

(+)= EYFP/no pTet

P1=plate 1 of pTet control ligation

P2=plate 2 of pTet control ligation

Lane

1. Ladder
2. E1-1
3. E1-2
4. E1-3
5. E1-4
6. E1-5
7. E2-1
8. E2-2
9. E2-3
10. E2-4
11. E2-5
12. +
13. –
14. P1-1
15. P1-2
16. P1-3
17. P1-4
18. P2-1
19. P2-2
20. P2-3

[![July 30-colonyPCR.png](/wiki/images/7/7c/July_30-colonyPCR.png)](/File%3AJuly_30-colonyPCR.png)

Completed the maxipreps of pBAD, TetR, pLacI, mRBS,sRBS and Lumazine 2. They were left at isopropanol precipitation.

Restriction Digest

dT-EcoRI & XbaI

pTet-SAP

pSB1A3-1 -EcoRI, Spe1

n-terminal tag-XbaI, PstI

Lumazine- XbaI, Spe1

pSB1A3-2- XbaI, PstI

C-terminal tag- EcoRI, Spe1

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | **dT** | *pTet* | pSB1A3-1 | **N-term** | *Lumazine* | pSB1A3-2 | **C-term** |
| DNA | **50 µL** | *20 µL* | 50 µL | **20 µL** | *20 µL* | 20 µL | **20 µL** |
| 10x buffer | **7 µL** | *3 µL* | 7 µL | **3 µL** | *3 µL* | 3 µL | **3 µL** |
| Enzyme 1 | **2 µL** | *1 µL* | 2 µL | **1 µL** | *1 µL* | 1 µL | **1 µL** |
| Enzyme 2 | **2 µL** | *0* | 2 µL | **1 µL** | *1 µL* | 1 µL | **1 µL** |
| milliQ water | **9 µL** | *6 µL* | 9 µL | **9 µL** | *9 µL* | 9 µL | **9 µL** |
| total | **70 µL** | *30 µL* | 70 µL | **30 µL** | *30 µL* | 30 µL | **30 µL** |

Prefix-N-terminal tag//gene
Gene//c-terminal-suffix

Ran a 1x agarose gel of PCR products from earlier.

Lane:

1. 1kb ladder
2. Control
3. CFP-phusion
4. EYFP-phusion
5. CFP-econo taq
6. EYFP-econo taq

[![July 30 C-term PCR.png](/wiki/images/b/b3/July_30_C-term_PCR.png)](/File%3AJuly_30_C-term_PCR.png)

The PCR worked

### July 31

**Mackenzie + Lisza**

Gel extraction of c-term tag, n-term tag and lumazine.

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## August

### Aug 4

**Roxanne and Kirsten**

DNA purification of the 2 pSB1A3 plasmids, dT and pTet using the gel extraction kit

Ran gel to check concentrations

* pTet 25ng/µL
* pSB1A3-2
* pSB1A3-1
* dT 180 ng/µL, 3318bp
* N-term 25 ng/µL, 507bp
* Lumazine 120 ng/µL, 861bp
* c-term 25 ng/µL, 55bp
* EYFP 878bp

Ligations of:

pTet-EYFP

Lumazine-dT

c-term-dT

Calculations:

EYFP: 3x(878bp [EYFP]/2211bp [pTet])x25ng

=29.78ng

=4µL EYFP:1 µL pTet

1 µL Lumazine: 1 µL dT

1 µL c-term:1 µL dt

|  |  |  |  |
| --- | --- | --- | --- |
|  | **pTet-EYFP** | *Lumazine-dT* | c-term-dT |
| milliQ water | **3.5 µL** | *6.5 µL* | 6.5 µL |
| 10x buffer | **1 µL** | *1 µL* | 1 µL |
| insert | **4 µL** | *1 µL* | 1 µL |
| Vector | **1 µL** | *1 µL* | 1 µL |
| Ligase | **0.5 µL** | *0.5 µL* | 0.5 µL |

**Megan/Mackenzie**

Gel extraction pSBIA3-1 and 2 (plasmid for biobrick parts)

Transformation: Tranformed to DH5alpha pBAD = promoter TetR= inverter. Added 2 micro liters of each ligation to DH5alpha. See July 14th Protocol

Restrictions of pBAD (SpeI/PstI), TetR (XbaI/PstI), PLacI (SpeI, PstI), sRBS (SpeI/ PstI), mRBS (SpeI/PstI). Used PCR tubes. Each tube contains: 5 micro liters MillQ water, 3 micro liters 10x Tango Buffer, 1 micro liter enzyme 1, 1 micro liter enzyme 2, 20 micro liters of DNA. Put all tubes in thermal controller at 37 degrees.

Roxanne,

Gel Extraction of pBAD, TetR, pLacI, sRBS, mRBS

Lengths:

pBAD:2287 bp

TetR: 902 bp

pLacI: 2279

sRBS: 2092 bp

mRBS: 2092 bp

Lane:

1. Ladder
2. TetR
3. pLacI
4. sRBS
5. mRBS
6. pBAD

[![Aug4.png](/wiki/images/9/99/Aug4.png)](/File%3AAug4.png)

### Aug 5th

**Kirsten and Fan**

Gel extraction of TetR, pLacI, sRBS, mRBS, pBAD according to Qiagen protocol.

Ran analytic gel

1µL Dye + 5µL DNA

6µL Ladder

Lane:

1. Ladder
2. TetR
3. pLacI
4. sRBS
5. mRBS
6. pBAD
7. pSB1A3-1
8. pSB1A3-2

[![Aug5.JPG](/wiki/images/2/2e/Aug5.JPG)](/File%3AAug5.JPG)

tetR concentration: 25ng/µL

everything else: 100ngµL

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### Aug 6

**Roxanne**

Set up the following ligations

pSB1A3-2+n-term tag and

pSB1A3-1+c-term tag

* 6.5µL milliQ water
* 1 µL 10x T4 buffer
* 1 µL insert
* 1 µL vector
* 0.5 µL T4 ligase

pSB1A3+lumazine

* 5.5µL milliQ water
* 1 µL 10x T4 buffer
* 2 µL insert
* 1 µL vector
* 0.5 µL T4 ligase

pBAD+tetR inverter

* 2.5µL milliQ water
* 1 µL 10x T4 buffer
* 5 µL insert
* 1 µL vector
* 0.5 µL T4 ligase

mRBS+n-term tag

* 6.5µL milliQ water
* 1 µL 10x T4 buffer
* 1 µL insert
* 1 µL vector
* 0.5 µL T4 ligase

Colony PCR of pTet-EYFP, lumazine-dT, c-term-dT

Picked 5 colonies of each construct, followed protocol of July 23.

E1-E5=EYFP colonies 1-5

E+=EYFP (no pTet)

L1-L5=lumazine-dT colonies 1-5

L+= lumazine (no dT)

C1-C5= c-term-dT colonies 1-5

C+= c-terminal (no dT)

-=no plasmid

**Lisza**

Mixed 10mM primers for: antisense suffix primer and FP N-term fusion primer

**Ashley, Mackenzie, Lisza**

Analytical gel of colony PCR’s (from earlier)

Volumes:

* 10 µL DNA
* 2 µL 6x loading
* 10 µL ladder

Lane

1. 1 kb ladder
2. –ve control
3. L1
4. L2
5. L3
6. L4
7. L5
8. L+
9. E1
10. E2
11. E3
12. E4
13. E5
14. E+
15. C1
16. C2
17. C3
18. C4
19. C5
20. C+

[![Aug 6.jpg](/wiki/images/1/12/Aug_6.jpg)](/File%3AAug_6.jpg)

Transformation of mRBS+n-term, pBAD+tetR, pSB1A3+lumazine, pSB1A3+c-term, pSB1A3+n-term

Negative control: water+25 µL DNA

Transformed according to iGEM protocol. Plated on ampicillin plates at 100µL and 400 µL volumes and incubated overnight.

PCR of EYFP and CFP according to protocol of July 30th.

### Aug 7

The transformations seemed to have all worked, although there are no colonies on plates marked pSBB1A3+N-term and several on the negative control plate. Speculation: tubes were switched or improper labeling.

Solution: Colony PCR

Lane:

1. Ladder (1kb)
2. Negative
3. TetR
4. pBAD+tetR colony 1
5. pBAD+tetR colony 2
6. pBAD+tetR colony 3
7. mRBS
8. mRBS+nterm colony 1
9. mRBS+nterm colony 2
10. mRBS+nterm colony 3
11. pSB1A3
12. c-term colony 1
13. c-term colony 2
14. c-term colony 3
15. N-term colony 1
16. N-term colony 2
17. N-term colony 3
18. Lumazine colony 1
19. Lumazine colony 2
20. Lumazine colony 3

Set up restriction digest of the preperative PCR of EYFP (c-term) and CFP(c-term) fusion proteins

DPNI, XbaI,PstI

DPNI, EcoRI, speI for the n-term proteins

Picked colonies of the transformed cells for pBAD-TetR, pSB1A3 (n-term), pSB1A3 (c-term), pSB1A3 (lumazine) and mRBS-Nterm

3x in ampicillin 5mL tubes.

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### Aug 11

**Roxanne**

Restricted all the minipreps with EcoRI to check the size of the plasmids prior to sending away for sequencing (10µL rxns)

Restricted:

* pSB-(n-term) w/ SpeI, PstI
* pSB-(c-term) w/ EcoRI, XbaI

in 30 µL rxns to prepare for fusion to CFP of EYFP

Setting up ligations of:

* pSB1A3 + EYFP (c-term)
* pSB1A3 + CFP (c-term)
* pSB1A3 + EYFP (n-term)
* pSB1A3 + CFP (n-term)

mmS6 gene was transformed into DH5α by Megan and Fan, set up a few cultures for minipreps.

### Aug 13

Set up restrictions of :

* mmS6 (EcoRI, SpeI)
* n-term CFP (EcoRI, SpeI)
* CheZ (EcoRI, SpeI)
* mRBS-N-term (SpeI, PstI)
* c-term-dt (EcoRI, XbaI)

Gel Extractions of:

* mms6
* cheZ

Using Qiagen bench top protocol

Set up ligations of:

* pSB1A3-1 + mmS6
* pSB1A3-1 + riboswitch
* pSB1A3-1 + n-term CFP
* pSB1A3-1 + n-term EYFP
* pSB1A3-2 + c-term CFP
* pSB1A3-2 + c-term EYFP
* mRBS + N-term CFP
* mRBS + N-term EYFP
* CheZ+dT
* mmS6 + dT

Ran a concentration gel of:

* c-term CFP
* c-term EYFP
* N-term CFP
* N-term EYFP
* c-term-dT
* mRBS-N-term

Concentrations:

* Riboswitch= 100ng/µL
* pSB1A3-1= 100ng/µL
* pSB1A3-2= 100ng/µL
* dT= 180ng/µL

Ran 1% agarose gel (with 2 wells taped together)

Lane:

1. 5µL of 1kb ladder
2. 20µL CheZ, 4µL dy

Ran at 110V for 30min

Gel extraction and purification of CheZ and mms6, mmS6 split into two tubes. Following Qiagen kit purification benchtop protocol.

Concentration Gel (1% agarose)

Lane:

1. 1 kb ladder (6µL)
2. N-term EYFP
3. C-N
4. Y-N
5. 4
6. 1
7. mmS6
8. C-term dT
9. mRBS+n-term

[![Concentration Gel Aug 13.jpg](/wiki/images/f/fc/Concentration_Gel_Aug_13.jpg)](/File%3AConcentration_Gel_Aug_13.jpg)

Only 9/18 transformations worked

Success:

* mmS6 in pSB1A3
* mmS6 in dT
* riboswitch in pSB1A3
* CheZ in dT
* CFP (no promoter)
* GFP gene
* pSB1A3 + n-term EFYP (1)
* pSB1A3 +n-term EYFP (2)
* fused c-EYFP

Added more ligase and will re-transform the following:

* Fused NEYFP (1)
* Fused NEYFP (2)
* Fused NCFP (1)
* Fused NCFP (2)
* Fused CEYFP
* Fused CCFP
* pSB1A3 +N-CFP (1)
* pSB1A3 +N-CFP (2)
* pSB1A3 +C-CFP (1)
* pSB1A3 +C-EYFP (1)

Transformed all of the above. Picked colonies of successes.

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### Aug 15th

The transformations didn’t work! Restart

Picked 3 colonies of cheZ-dT

1 of mmS6+dT (for mini and maxiprep)

3 of fused C-EYFP

3 of pSB1A3-mmS6

3 of riboswitch in pSB1A3

3 of GFP gene

3 of CFP (no promoter)

2 of the riboswitch and all of the N-EYFP in pSB1A3 turned red suggesting that the RFP reporter was religated in.

Will miniprep the “good” cells and restrict with EcoRI to check for size

Glycerol stock.

### Aug 18

**Megan and Ashley**

Pelleted down cells (small samples and large samples) picked from transformations to prep for mini-preps.

Add 750 x2 micro liters of your small sample solution to centrifuge tube, Add 50 mL of large sample to large centrifuge tubes. Balanced the Centrifuge and ran for 2 minutes at 13000 rpm. Dump out Supernatant/ leave pellet. Add more sample, centrifuge, and remove more supernatant. Add remainder of sample, centrifuge, remove supernatant. Left samples in -20 degree freezer.

### Aug 19

**Kirsten and Mackenzie**

Maxiprep of fused C-EYFP and mmS6-dT

See protocol for July 13th.

### Aug 20

**Fan and Mackenzie**

Miniprep for:

* cheZ-dT
* mmS6-dT
* PSB1A3-mmS6
* GFP
* CFP no promo
* Fused C-EYFP
* Riboswitch (2,4,7,8,9)

Restriction digest with EcoRI

* 10µL DNA
* 2 µL 10x buffer tango
* 4 µL EcoRI
* Total volume = 20 µL

37°C for 1 hour.

Need to run agarose for all restriction digested samples and DNA samples

**Roxanne, Mackenzie**

Running a gel of restricted minipreps

Checking mini prep concentration with UV spec

Lane

1. 1 kb ladder
2. cheZ-dT 1 (78 µg/mL)
3. cheZ-dT 2 (80 µg/mL)
4. cheZ-dT 3 (106 µg/mL)
5. mmS6-dT 1 (82 µg/mL)
6. mmS6-dT 2(91 µg/mL)
7. mmS6-dT 3 (144 µg/mL))
8. pSB-mmS6 1 (125 µg/mL)
9. pSB-mmS6 2(88 µg/mL)
10. pSB-mmS6 3(135 µg/mL)
11. GFP 1(-------)
12. GFP 2 (108 µg/mL))
13. GFP 3(98 µg/mL)
14. CFP no promo 1(----------)
15. CFP no promo 2 (134 µg/mL)
16. Fused C-EYFP 1 (49 µg/mL)
17. Fused C-EYFP 2 (174 µg/mL)
18. Fused-CEYFP 3 (75 µg/mL)
19. 1 kb ladder)

[![Aug 20, big gel.jpg](/wiki/images/9/9a/Aug_20%2C_big_gel.jpg)](/File%3AAug_20%2C_big_gel.jpg)

Lane

1. 6 µL 1 kb ladder
2. 6 µL pSB1A3
3. Riboswitch 2 (152 µg/mL)
4. Riboswitch 4 (81 µg/mL)
5. Riboswitch 7 (68 µg/mL)
6. Riboswitch 8 (112 µg/mL)
7. Riboswitch 9 (88 µg/mL)
8. 6 µL 1 kb ladder

[![Aug 20, little gel.jpg](/wiki/images/0/0b/Aug_20%2C_little_gel.jpg)](/File%3AAug_20%2C_little_gel.jpg)

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### Aug 25

**Roxanne**

Set up restrictions

1,4,7 are c-term

2,5,8 are n-term

1,2=EYFP

4,5=CFP

7,8=ECFP

* Fusion 1,4,7 restrict with DPN1, EcoRI, SpeI
* Fusion 1,4,7 restrict with DPN1, XbaI, PstI
* Fusion 2,5,8 restrict with DPN1, EcoRI, SpeI
* Fusion 2,5,8, restrict with DPN1, XbaI, PstI
* Riboswitch restrict with SpeI, PstI
* CheZ-dT restrict with XbaI, PstI
* GFP restrict with EcoRI, SpeI

* 4 µL milliQ water
* 2 µL 10x Tango buffer
* 12 µL DNA
* 1 µL E1
* 1 µL E1

Set up maxipreps for:

* Lumazine-dT
* mmS6-dT

Ligations:

* Fusion 1 + (c-term)-dT
* Fusion 4 + (c-term)-dT
* Fusion 7 + (c-term)-dT
* Fusion 1 + pSB1A3-2
* Fusion 4 + pSB1A3-2
* Fusion 7 + pSB1A3-2
* Fusion 2 + mRBS (n-term)
* Fusion 5 + mRBS (n-term)
* Fusion 8 + mRBS (n-term)
* Fusion 2 +pSB1A3-1
* Fusion 5 +pSB1A3-1
* Fusion 8 +pSB1A3-1
* Riboswitch +CheZ-dT
* GFP + dT
* sRBS + lumazine-dT
* sRBS + mmS6-dT

### Aug 26

**Lisza**

Deactivated to reactions @ 65° for 15 min

**Roxanne**

Set up liquid cultures of:

* mmS6 in pSB1A3
* mmS6-dT
* lumazine in pSB1A3
* lumazin-dT
* c-term in pSB1A3
* c-term-dT
* n-term in pSB1A3
* mRBS-(n-term)
* pTet-EYFP
* pBAD-TetR
* riboswitch in pSB1A3

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### Aug 27

**Fan**

Miniprep of the O/N culture

[DNA] unknown

DNA was eluted from the spin column in 50mL milliQ water

* 20µL of each DNA sample was sent to iGEM (MIT)

**Kirsten**

Gel extraction of GFP, mmS6-dT,CheZ-dT

Purified riboswitch, sRBS, c-term-dT, N-term-mRBS. Eluted with 50µL of EB

Into -20°C freezer.

10µL ligations of GFP+dT, sRBS+mmS6-dT and riboswitch+cheZ. Left at room temp for 2 hours until transformation.

**Alix**

Transformations of ligations following iGEM protocol

### Aug 31

**Roxanne**

PCR of CFP, ECFP, EYFP w/ n-term prefix or c-term suffix

20µL Restrictions of:

* pLacI with SpeI,PstI
* pStrong with SpeI,PstI
* Lumazine-dT with XbaI ,PstI

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## September

### September 3

**Roxanne, Ashley, Kirsten**

Made 1.5L agar LB media according to lab protocol. Roxanne is autoclaving tomorrow.

Pick 3 lumazine-dT colonies from glycerol stocks to grow overnight

Restriction Digest

Reaction 1

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | **Ribo-cheZ-dT** | *GFP-dT* | sRBS-mmS6 | **Mr. Gene mmS6** |
| milliQ water | **5 µL** | *5 µL* | 5 µL | **5 µL** |
| Buffer tango | **3 µL** | *3 µL* | 3 µL | **3 µL** |
| DNA | **20 µL** | *20 µL* | 20 µL | **20 µL** |
| XbaI | **1 µL** | *1 µL* | 1 µL | **1 µL** |
| PstI | **1 µL** | *1 µL* | 1 µL | **1 µL** |
| total | **30 µL** | *30 µL* | 30 µL | **30 µL** |

Reaction 2

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | **Ribo-cheZ-dT** | *GFP-dT* | sRBS-mmS6 | **Mr. Gene mmS6** |
| milliQ water | **3 µL** | *3 µL* | 3 µL | **3 µL** |
| Buffer tango | **1 µL** | *1 µL* | 1 µL | **1 µL** |
| DNA | **5 µL** | *5 µL* | 5 µL | **5 µL** |
| EcoRI | **1 µL** | *1 µL* | 1 µL | **1 µL** |
| total | **10 µL** | *10 µL* | 10 µL | **10 µL** |

Into HWB at 37°C for 2 hours

Gel Electrophoresis (concentration):

Lane

1. 1kb ladder
2. ribo-cheZ-dT (EcoRI)
3. GFP-dT (EcoRI)
4. sRBS-mmS6-dT (EcoRI)
5. NEYFP (XbaI,PstI)
6. NEYFP (ecoRI, speI)
7. NCFP (ecoRI, speI)
8. NECFP (xbaI,pstI)
9. CCFP(ecoRI,speI)
10. CECFP(ecoRI,speI)
11. CCFP (notI,pstI)
12. NEYFP (ecoRI,speI)
13. CECFP (xbaI)
14. CEYFP(xbaI,pstI)
15. NCFP (xbaI,pstI)
16. CEYFP(xbaI)
17. pLacI
18. pStrong
19. 1kb ladder

5µL sample, 1 µL loading dye (6x)
6 µL ladder

[![September 3 concentration gel.png](/wiki/images/8/81/September_3_concentration_gel.png)](/File%3ASeptember_3_concentration_gel.png)

Ran at 100V for 1 hour

Concentrations good!

**Alix**

Gel extraction of:

sRBS-mmS6-dT

riboswitch-cheZ-dT

GFP-dT

mmS6

Following Qiagen benchtop protocol

### September 4

**Ashley**

Ligation of:

* C-term-dT +C-CFP #2
* C-term-dT +C-ECFP #2
* C-term-dT + C-EYFP #2
* pSB1A3-2+C-CFP#1
* pSB1A3-2+C-ECFP#1
* pSB1A3-2+C-EYFP#1
* mRBS-N-term +N-CFP#1
* mRBS-N-term +N-EYFP#1
* mRBS-N-term +N-ECFP#1
* pSB1A3-1+N-CFP#2
* pSB1A3-1+N-ECFP#2
* pSB1A3-1+N-EYFP#2

Reaction

|  |  |  |
| --- | --- | --- |
|  | **Control** | All reactions |
| milliQ water | **7.5 µL** | 4.5 µL |
| 10x buffer T4 | **1 µL** | 1 µL |
| Vector | **1 µL** | 1 µL |
| Insert | **0 µL** | 3 µL |
| T4 DNA ligase | **0.5 µL** | 0.5 µL |
| total | **10 µL** | 10 µL |

Restriction Digest

|  |  |  |
| --- | --- | --- |
|  | Control (no enzyme) | **Lumazine-dT (1)** |
| milliQ water | 2.4 µL | **0.4 µL** |
| 10x buffer Tango | 1.6 µL | **1.6 µL** |
| DNA | 16 µL | **16 µL** |
| XbaI | 0 µL | **1 µL** |
| PstI | 0 µL | **1 µL** |
| total | 20 µL | **20 µL** |

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### September 8

**Ashley**

Made 1.5L LB agar according to protocol

Made 300mL LB broth according to protocol

Gel Electrophoresis (for extraction)

5 µL ladder

5 µL sample, 1 µL dye

Lane

1. 1 kb ladder
2. empty^
3. empty
4. lumazine-dT (XbaI, PstI) 600bp

^there was a control (no enzyme) but not enough DNA to load

Ran at 1000V for one hour

Gel extraction according to Qiagen benchtop protocol

### Sept 10

**Ashley, Kirsten**

Ran 1% agarose

Lane

1. 1 kb ladder
2. C-term-dT + C-EYFP #2
3. pSB1A3-1+N-CFP#2
4. pSB1A3-1+N-ECFP#2
5. lumazine+sRBS
6. pSB1A3-2+C-EYFP#1
7. pSB1A3-1+N-EYFP#2
8. mRBS-N-term +N-CFP#1
9. mmS6+pSB1A3-1
10. GFP-dT
11. mmS6-dT
12. pSB1A3-2+C-ECFP#1
13. mRBS-N-term +N-ECFP#1
14. mRBS-N-term +N-EYFP#1
15. C-term-dT +C-CFP #2
16. C-term-dT +C-ECFP #2
17. pSB1A3-2+C-CFP#1

3 µL sample + 0.5 µL dye

6 µL 1 kb ladder

**Mackenzie**

Transformation into DH5α

* sRBS-lumazine-dT (km)
* mmS6-dT (km)
* pSB1A3+mmS6
* mRBS-N-term +N-ECFP#1
* mRBS-N-term +N-EYFP#1
* C-term-dT +C-ECFP #2
* C-term-dT + C-EYFP #2
* pSB1A3-2+C-ECFP#1
* pSB1A3-2+C-EYFP#1

The following were not transformed due to lack of competent cells:

* pSB1A3-1+N-ECFP#2
* pSB1A3-1+N-EYFP#2
* GFP-dT

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### September 14

**Ashley**

PCR of EYFP, CFP and ECFP

See Aug 6 for volumes

CFP(1)=prefix primer and antisense C-term suffix

CFP(2)=N-term prefix and antisense suffix

EYFP(1)=prefix primer and antisense C-term suffix

EYFP(2)=N-term prefix and antisense suffix

ECFP(1)=prefix primer and antisense C-term suffix

ECFP(2)=N-term prefix and antisense suffix

Cycle: tail67

### Sept 16

**Ashley**

Miniprep of mmS6-dT 1,2 and 3. Used Qiagen benchtop protocol and kit. Eluted with 50 µL buffer EB. Put into -20°C freezer orange tray.

PCR Purification of restriction digest of PCR products.

### September 24

**Ashley, Kirsten**

1% gel electrophoresis
6uL ladder (1kb)
5uL sample +1 uL 6x loading dye
100V, 40min

Lane

1. 1kb Ladder
2. C-CFP-1
3. C-ECFP-1
4. N-EYFP-2
5. N-CFP-2
6. N-ECFP-2
7. C-EYFP-1
8. CFP-1 Purified PCR
9. ECFP-1 Purified PCR
10. EYFP-2 Purified PCR
11. CFP-2 Purified PCR
12. ECFP-1 Purified PCR
13. EYFP-1 Purified PCR
14. C-CFP(1)
15. ECFP(1)
16. C-EYFP(1)
17. N-CFP(2)
18. N-EYCFP(2)
19. N-EYFP(2)
20. 1KB LADDER

[![Sept 24 - control for PCR.png](/wiki/images/9/94/Sept_24_-_control_for_PCR.png)](/File%3ASept_24_-_control_for_PCR.png)

Gel was run to check for DNAses, found that DPN-1 had DNAses.

* [Home](https://2009.igem.org/Team%3ALethbridge)
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### Sept 25

1% agarose gel 100V, 40min

Lane:

1. 1kb ladder
2. mms6(EcoRI,SpeI)
3. pLacI (SpeI, PstI)
4. Lumazine-dT

Only pLacI success, gell extracted and eluted with 50uL buffer EB.

Miniprep of:

* 3x mmS6-dT
* 3x pSB1A3-mmS6
* 2x cheZ-dT
* 1x riboswitch-cheZ-dT
* 2x GFP-dT
* 3x C-EYFP-dT

Restricted all of the above with EcoRI

* 8uL DNA
* 1uL 10x buffer tango
* 1uL EcoRI

for 1 hour.

Restriction of fusion proteins with either EcoRI,SpeI or XbaI,PstI

* Heat inactivated a 60C for 10min

Ran on a gel (1% agarose)100V,40min

Lane:

1. 1kb ladder
2. pSB-mmS6 #1
3. pSB-mmS6 #2
4. pSB-mmS6 #3
5. mms6-dT #1
6. mms6-dT #2
7. mms6-dT #3
8. EYFP C-term-dT #1
9. EYFP C-term-dT #2
10. EYFP C-term-dT #3
11. GFP-dT#1
12. GFP-dT#2
13. Riboswitch-cheZ-dT
14. cheZ-dT#1
15. cheZ-dT#2
16. C-CFP (EcoRI,SpeI)
17. C-CFP (XbaI,PstI)
18. C-ECFP (EcoRI,SpeI)
19. C-ECFP (XbaI,PstI)
20. 1kb laddder

[![Sept 25-first lanes.png](/wiki/images/5/59/Sept_25-first_lanes.png)](/File%3ASept_25-first_lanes.png)

[![Sept 25 far lanes.png](/wiki/images/0/07/Sept_25_far_lanes.png)](/File%3ASept_25_far_lanes.png)

* Restriction of Fusion proteins with Dpn1
* Heat inactivated a 60C for 10min

Lane:

1. 1kB ladder
2. N-ECFP
3. N-ECFP
4. N-EYFP
5. E-EYFP
6. N-CFP
7. N-CFP
8. C-EYFP
9. C-EYFP
10. 1kB ladder

Restriction of Lumazine-dT

* 30uL DNA
* 4uL 10x buffer
* 3uL EcoRI
* 3uL SpeI

Setup Liquid culture of the Mr. Gene mmS6 x3

### September 28

**Ashley**

1% agarose gel

* 5uL DNA+1uL 6x loading dye
* 6uL ladder (1kb)

Lane:

1. 1kb ladder
2. N-ECFP (XbaI,PstI)
3. N-ECFP (EcoRI, SpeI)
4. N-EYFP(XbaI,PstI)
5. N-EYFP(EcoRI, SpeI)
6. N-CFP(XbaI,PstI)
7. N-CFP(EcoRI, SpeI)
8. C-EYFP(XbaI,PstI)
9. C-EYFP(EcoRI, SpeI)
10. 1kb ladder

Ran at 100V for 40 min

[![September 28th.jpg](/wiki/images/9/9f/September_28th.jpg)](/File%3ASeptember_28th.jpg)

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

## October

### October 1

**Roxanne**
Gel for gel extraction

Restriction
Ribo-Chez-dT

* 2x CheZ-dT
* 2x GFP-dT
* 3x C-EYFP-dT

Ligation

* C-ECFP-dT
* C-CFP-dT
* C-EYFP-dT
* mRBS-N-ECFP
* mRBS-N-EYFP
* pSB-C-EFP
* pSB-C-EYFP
* pSB-C-CFP
* pSB-N-ECFP
* pSB-N-EYFP

|  |  |  |
| --- | --- | --- |
| Tube | **Tube + Gel** | Gel |
| 1.1226g | **1.4931g** | 0.2671g |
| 1.012g | **1.2302g** | 0.1290g |

Eluted with 50uL buffer EB

Ligation

|  |  |  |
| --- | --- | --- |
|  | **1:3** | 2:6 |
| lum-dT | **3uL** | 6uL |
| SRBS | **1uL** | 2uL |
| Ligase Buffer | **1uL** | 2uL |
| Water | 4.5uL | 9uL |
| Ligase | 0.5uL | 1uL |

### Oct 2

Pick Colonies:

* sRBS + Lumazine-dT
* mRBS + N-term FPs
* C-term FPs + dT
* pSB + N-term FPs
* pSB + N-term FPs

Gel Extraction:

* Riboswitch-cheZ-dT
* 2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
* 3x C-EYFP-dT
* 2x GFP-dT

Liquid Culture:

* Riboswitch-cheZ-dT
* 2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
* 3x C-EYFP-dT
* 2x GFP-dT

Miniprep:

* Mr. Gene mms6

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### Oct 3

Miniprep:

* sRBS + Lumazine-dT
* mRBS + N-term FPs
* C-term FPs + dT
* pSB + N-term FPs
* pSB + N-term FPs
* sRBS + Lumazine-dT

Restriction:

Analytic and Prep:

* mRBS + N-term FPs
* C-term FPs + dT
* pSB + N-term FPs
* pSB + N-term FPs

Analytic only:

* Mr. Gene mms6

Prep only:

* pBAD-TetR

### Oct 5

Gel extraction:

* sRBS + Lumazine-dT
* mRBS + N-term FPs
* C-term FPs + dT
* pSB + N-term FPs
* pSB + N-term FPs

Ligation:

* pStrong + Riboswitch-cheZ-dT
* Riboswitch-GFP-dT
* pLac + sRBS-Lumazine-dT
* pBad-TetR + C-term FPs
* pBad-TetR + N-term FPs
* pTetR + N-term

Transformation:

* pLac + sRBS-Lumazine-dT
* pBad-TetR + C-term FPs
* pBad-TetR + N-term FPs
* pTetR + N-term ECFP
* pStrong + Riboswitch-cheZ-dT
* Riboswitch-GFP-dT

Sequencing:

* sRBS + Lumazine-dT
* mRBS + N-term FPs
* C-term FPs + dT
* pSB + N-term FPs
* pSB + N-term FPs
* Mr. Gene mms6

### Tues Oct 6

Pick Colonies:

* pLac + sRBS-Lumazine-dT
* pBad-TetR + C-term FPs
* pBad-TetR + N-term FPs
* pTetR + N-term ECFP
* pStrong + Riboswitch-cheZ-dT
* Riboswitch-GFP-dT

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### Wed Oct 7

Miniprep:

* pLac + sRBS-Lumazine-dT
* pBad-TetR + C-term FPs
* pBad-TetR + N-term FPs
* pTetR + C-term ECFP
* pTetR + N-term ECFP
* pStrong + Riboswitch-cheZ-dT
* Riboswitch-GFP-dT

Analytic Restriction:

* pLac + sRBS-Lumazine-dT
* pBad-TetR + C-term FPs
* pBad-TetR + N-term FPs
* pTetR + N-term ECFP
* pStrong + Riboswitch-cheZ-dT
* Riboswitch-GFP-dT

Prep Restriction:

* Riboswitch-GFP-dT

### Thurs Oct 8

Gel Extraction:

* Riboswitch-GFP-dT

Ligation:

* pStrong + Riboswitch-GFP-dT

Transformation:

* pStrong + Riboswitch-GFP-dT

Sequencing:

* pLac-sRBS-Lumazine-dT
* pBad-TetR-C-term FPs
* pBad-TetR-N-term FPs
* pTetR-N-term ECFP
* pStrong-Riboswitch-cheZ-dT
* Riboswitch-GFP-dT

Maxiprep:

* pLac-sRBS-Lumazine-dT
* pBad-TetR-C-term FPs
* pBad-TetR-N-term FPs
* pTetR-N-term ECFP
* pStrong-Riboswitch-GFP-dT

Restriction:

* pLac-sRBS-Lumazine-

Analytic and Prep:

* pBad-TetR-C-term FPs
* pBad-TetR-N-term FPs
* pTetR-N-term ECFP
* C-term CFP (has pTetR)

Analytic Only:

* pStrong-Riboswitch-GFP-dT

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### Fri Oct 9

Overexpression:

* pLac + sRBS-Lumazine-dT

Gel Extraction

* pLac-sRBS-Lumazine-dT
* pBad-TetR-C-term FPs
* pBad-TetR-N-term FPs
* pTetR-N-term ECFP
* C-term CFP (has pTetR)

Motility Media

### Oct 12

Ligation:

* pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
* pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Transformation:

* pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
* pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Plate Testing:

* pStrong-Riboswitch-GFP-dT

Motility Test:

* pStrong + Riboswitch-cheZ-dT

### Oct 13

Pick Colonies

* pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
* pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Analysis of Plate and Motility Tests

* [Home](https://2009.igem.org/Team%3ALethbridge)
* [Top of Page](https://2009.igem.org/Team%3ALethbridge/Notebook)

### Wed Oct 14

Miniprep:

* pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
* pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Restriction:

* pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
* pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

### Thurs Oct 15

Gel Extraction:

* pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
* pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Ligation:

* pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
* pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Transformation:

* pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
* pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Expression Testing:

* pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
* pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

### Fri Oct 16

Pick Colonies:

* pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
* pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

### Sat Oct 17

Maxiprep:

* pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
* pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

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