iGEM 2016 — Team Emory

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# Team:Emory/Notebook

Team:Emory iGEM 2016

* [HOME](https://2016.igem.org/Team%3AEmory)
* PROJECT
  + [Description](https://2016.igem.org/Team%3AEmory/Description)
  + [Experiments](https://2016.igem.org/Team%3AEmory/Experiments)
  + [Proof of Concept](https://2016.igem.org/Team%3AEmory/Proof)
  + [Results](https://2016.igem.org/Team%3AEmory/Results)
  + [Notebook](https://2016.igem.org/Team%3AEmory/Notebook)
* [PARTS](https://2016.igem.org/Team%3AEmory/Basic_Part)
* [SAFETY](https://2016.igem.org/Team%3AEmory/Safety)
* [COMMUNITY INVOLVEMENT](https://2016.igem.org/Team%3AEmory/HP/Silver)* [COLLABORATIONS](https://2016.igem.org/Team%3AEmory/Collaborations)
  * [TEAM](https://2016.igem.org/Team%3AEmory/Team)
  * [ATTRIBUTIONS](https://2016.igem.org/Team%3AEmory/Attributions)

# NOTEBOOK

We've recorded all of our achievements and blunders along the way, so you can follow our thought process as we overcame challenges and moved forward in our research.

#### [EXPERIMENT iG1: Subcloning pWH1266 plasmid origin into pSB1C3](#collapse1)

**Purpose:** to subclone the pWH1266 origin of replication (for Acinetobacter plasmids) and several selectable markers into the iGEM standard plasmid, pSB1C3.

**Procedure:**

**1a. plasmid prep (see imc229e)**

229e. more plasmid preps

Parts to subclone into pSB1C3 (standard iGEM)

40 mL LB-chl

1. imc229c3 Plac-lacO-rfp-TT-pSB1C3 (Mach1)

6 mL LB-kan

2. imc104L2.2 /pWH1266 ori-IMBB-kanR (pIM1310, InvaF')

6 mL LB-amp

6 mL LB-amp

3. imc212t3.2 T1-lacI-PT5-rpmH-sfBFP-pIDT (pIM1610, Mach1)

4. BS57h4.1 tetR-PtetA-tetM-pUC57-mini (pIM1842, Mach1)

5. imc181r4.1 Ptac-lacOc-aph(3')-VIa-IMBB (pIM216, InvaF')

6. imc203d6.1 Ptac-lacOc-ampC ADC-33-pMA (pIM1406, Mach1)

37 deg rotator 6/17/2016 6:28 PM IM

QIAgen miniprep

eluted 50uL EB

Take 3 concentration measurement

1. imc229c3 Plac-lacO-rfp-TT-pSB1C3 41.994 ng/uL, 77.387, 68.031

2. imc104L2.2 pWH1266 ori-IMBB-kanR (pIM1310, InvaF')

3. imc212t3.2 T1-lacI-PT5-rpmH-sfBFP-pIDT 56.994

4. BS57h4.1 tetR-PtetA-tetM-pUC57-mini 36.411

5. imc181r4.1 Ptac-lacOc-aph(3')-VIa-IMBB (pIM216, InvaF')

6. imc203d6.1 Ptac-lacOc-ampC ADC-33-pMA 30.006

**1b. restriction digest to subclone T1-lacI-PT5-rpmH-sfBFP into pSB1C3**

vector a: desire 2047 bp, not 1092

30 uL imc229c3 Plac-lacO-rfp-TT-pSB1C3

8 uL 10x NEB 2.1

39 uL ddH2O

1 uL EcoRI-HF = 20 units

2 uL SpeI = 20 units

---

80 uL

insert b: desire 2808 bp, not 1336 or 790 bp

30 uL imc212t3.2 T1-lacI-PT5-rpmH-sfBFP-pIDT

8 uL 10x NEB 2.1

38 uL ddH2O

1 uL EcoRI-HF = 20 units

2 uL SpeI = 20 units

1 uL ClaI = 10 units

---

80 uL

Gel lanes:

2log ladder

1a1a pSB1C3

1b VA (cut with enzymes)

1A3 sFBFP

1b1b (bands too small)

One well 0.8% LE agarose in TAE minigel

QIAcube gel extraction of vector only (and positive control insert)

Eluted in 50 uL EB

Michael/Maruf

**1c. restriction digest to subclone tetR-PtetA-tetM into pSB1C3**

vector c: desire 2047 bp, not 1092

30 uL imc229c3 Plac-lacO-rfp-TT-pSB1C3

8 uL 10x NEB 2.1

39 uL ddH2O

1 uL EcoRI-HF = 20 units

2 uL SpeI = 20 units

---

80 uL

insert d: desire 2658 bp, not 1855 bp

40 uL BS57h4.1 tetR-PtetA-tetM-pUC57-mini

8 uL 10x NEB 2.1

29 uL ddH2O

1 uL EcoRI-HF = 20 units

2 uL SpeI = 20 units

---

80 uL

Gel lane:

1c vector c cut

1a-1b pSB1c3

1a-4 tetM (one band instead of two)

1c vector c cut (replicated)

1c insert d

2-Log DNA Ladder

One well 0.8% LE agarose in TAE minigel

QIAcube gel extraction of vector only (and positive control insert)

Eluted in 50 uL EB

**1d. restriction digest to subclone Ptac-lacOc-ampC ADC-33 into pSB1C3**

vector c: desire 2047 bp, not 1092

30 uL imc229c3 Plac-lacO-rfp-TT-pSB1C3

8 uL 10x NEB 2.1

39 uL ddH2O

1 uL EcoRI-HF = 20 units

2 uL SpeI = 20 units

---

80 uL

insert d: desire 1255 bp, not 2390 bp

40 uL imc203d6.1 Ptac-lacOc-ampC ADC-33-pMA

8 uL 10x NEB 2.1

29 uL ddH2O

1 uL EcoRI-HF = 20 units

2 uL SpeI = 20 units

---

80 uL

37 deg 6/18/2016 5:34 PM

Accidentally put digests into freezer

Experiment stopped

Sujith

**1e. restriction digests to subclone pWH1266 and Ptac-lacOc-aph(3')-VIa into pSB1C3**

(= imc229f)

vector g: desire 2047 bp, not 1092

15 uL imc229e1/229c3 Plac-lacO-rfp-TT-pSB1C3

8 uL 10x NEB 2.1

54 uL ddH2O

1 uL EcoRI-HF = 20 units

2 uL SpeI = 20 units

---

80 uL

insert h: desire 1374 bp, not 2077 bp

15 uL imc229e2/104L2.2 pWH1266 ori-IMBB-kanR

8 uL 10x NEB 2.1

54 uL ddH2O

1 uL EcoRI-HF = 20 units

2 uL SpeI = 20 units

---

80 uL

insert i: desire 954 bp, not 2315 bp

69 uL imc229e5/181r4.1 Ptac-lacOc-aph(3')-VIa-IMBB

8 uL 10x NEB 2.1

1 uL EcoRI-HF = 20 units

2 uL SpeI = 20 units

---

80 uL

37 deg 6/19/2016 9:52 AM

0.8% LE agarose minigel 1 in TAE

1. 1 uL imc229e1/229c3 Plac-lacO-rfp-TT-pSB1C3 uncut

2. 1 uL Plac-lacO-rfp-TT-pSB1C3 EcoRI/SpeI

3. 1 uL imc229e2/104L2.2 pWH1266 ori-IMBB-kanR uncut

4. 1 uL pWH1266 ori-IMBB-kanR EcoRI/SpeI

5. 1 uL imc229e5/181r4.1 Ptac-lacOc-aph(3')-VIa-IMBB uncut

6. 1 uL Ptac-lacOc-aph(3')-VIa-IMBB EcoRI/SpeI

7. 250 ng lambda HindIII

one well 0.8% LE agarose minigels in TAE

QIAcube gel extraction

eluted in 50 uL EB

Take 3

1. imc229f2 pWH1266 ori insert 13.31 ng/uL

2. imc229f3 Ptac-lacOc-aph(3')-VIa insert 2.685 ng/uL

3. iG1b2 positive control insert 5.073 ng/uL

4. iG1b1 pSB1C3 vector 6.383 ng/uL

5. iG1c1 pSB1C3 vector 12.628 ng/uL

6. iG1c2 positive control insert 1.7864 ng/uL

3. imc229f1 positive control insert 6.481 ng/uL

4. imc229f2 pSB1C3 vector 5.506 ng/uL

0.8% LE agarose minigel 1 in TAE ???

1. 5 uL iG1C1 pSB1C3 vector

2. 5 uL iG1C2 post cont ins

3. 5 uL imc229f1 pos cont ins

4. 5 ul imc229f1 pSB1C3 vector

5. 250 ng lambda HindIII

0.8% LE agarose minigel 2 in TAE

1 = 250 ng lambda HindIII

1 = 5 uL imc229f2 pWH1266 ori insert ~25 ng/uL

2 = 5 uL imc229f3 Ptac-lacOc-aph(3')-VIa insert ~2 ng/uL

3 = 5 uL iG1B1 post cont ins ~2 ng/uL

4 = 5 ul iG1B1 pSB1C3 vector ~15 ng/uL

1fgh. Ligation of pWH1266 ori and Ptac-lacOc-aph(3')-VIa inserts into pSB1C3 vector

F = Cyrillus

G = Sujith

H = Ichiro.

20 fmoles pSB1C3 vector a = 13 ng/kb x 2 kb = 26 ng = 2 uL

20 fmoles pWH1266 ori insert b = 13 ng/kb x 1.4 kb = 18 ng = 2 uL

20 fmoles Ptac-lacOc-aph(3')-VIa insert c = 13 ng/kb x 1 kb = 13 ng = 5 uL

20 fmoles positive control Plac-lacO-rfp-TT insert d = 13 ng/kb x 1.1 kb = 15 ng = 2 uL

F = Cyrillus/Jasmine

G = Sujith/Maruf

H = Ichiro, 37 deg 6/26/16 3:50 p.m.

Results: 6/27/16

None of these worked

1G1: 0 cfu/100 uL

1G2: 0 cfu/100 uL

1G3: 2 cfu/100 uL

1G4: 3 cfu/100 uL

1G5: 3 cfu/100 uL

1G6: 3 cfu/100 uL (one red)

1j. Re-streak some colonies on LB-kanamycin plates

6/28/16 5:16 p.m.

None formed colonies except imc181r4.1 Ptac-lacOc-aph(3')-VIa-IMBB control. This suggests that the pSB1C3 vector can somehow recircularize without forming the parental construct.

#### [EXPERIMENT iG2-iG5: Create Shuttle Vectors for A. Baylyi](#collapse2)

**Experiment iG2**

**Purpose:** to subclone the Ptac-lacOc-R67-DHFR insert into the pWH1266 ori-pSB1C3 to create A. baylyi-E. coli shuttle vector pWH1266 ori-Ptac-lacOc-R67-DHFR-pBS1C3 (trimethoprim resistance)

IV. Procedure

2a. Plasmid prep

6 mL LB-chl

1. imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926)

6 mL LB-amp

2. alw4m1.1 Ptac-lacOc-R67\_DHFR-pMA (pIM1651, InvaF')

37 deg rotator 7/11/2016 6:24 AM

QIAprep miniprep

Eluted each with 100uL EB

Gel filtration with G-50

Take3 concentration measurement

**Experiment iG3**

**Purpose** to subclone the Ptac-lacOc-aph(3')-VIa assembly insert into the pWH1266 ori-pSB1C3 to create A. baylyi-E. coli shuttle vector pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

**Procedure**

3a. Plasmid Prep

6 mL LB-chl

1. imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926)

2. imc229j4.1 are Ptac-lacOc-aph(3')-VIa-pSB1C3 (pIM1927)

37 deg rotator 7/6/2016 2:46 PM

Used Qiacube miniprep protocol. Eluted with 60uL of EB buffer.

7/7/2016 11:10 AM

3b. Gel filtration

1. imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926)

25.524 ng/uL

2. imc229j4.1 are Ptac-lacOc-aph(3')-VIa-pSB1C3 (pIM1927)

39.579 ng/uL

4c. Restriction digest and concentration measurement

1. imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926)

50uL volume

1uL Spe1

1uL Pst1

5uL NEB 10X NEB 2.1

39uL pWH1266 ori-pSB1C3

4uL ddH20

2. imc229j4.1 are Ptac-lacOc-aph(3')-VIa-pSB1C3 (pIM1927)

50uL volume

1uL Xba

1uL Pst1

5uL NEB 10X

25uL Ptac-lacOc-aph(3')-VIa-pSB1C3

18uL ddH20

Samples incubating in 37degree room

7/9/2016 4:58 PM

Ran gel of uncut and cut plasmids

Lane - Sample

1 - DNA Ladder

2 - Uncut pWH1266 ori-pSB1C3

3 - Uncut Ptac-lacOc-aph(3')-VIa-pSB1C3

4 - Cut pWH1266 ori-pSB1C3

5 - Cut Ptac-lacOc-aph(3')-VIa-pSB1C3

7/10/2016 5:15 PM

3d. Gel isolation and extraction

1. imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926)

5.933 ng/uL

2. imc229j4.1 are Ptac-lacOc-aph(3')-VIa-pSB1C3 (pIM1927)

2.131 ng/uL

3e. Ligation

Very little yield in gel extraction, 20uL final volume for ligation is not feasible.

Reaction 4.1

9uL 5X T4 DNA ligase buffer

5uL vector DNA

40uL insert DNA

1uL T4 DNA

Reaction 4.2- Control

9uL 5X T4 DNA ligase buffer

9uL 5X T4 DNA ligase buffer

5uL vector DNA

1uL T4 DNA 7/26/2016 2:58 PM

4f. Competent cell transformation

1. 5.1 pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

2. 5.2 Control pWH1266 ori-pSB1C3

3. 5.1A pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

4. 5.2A Control pWH1266 ori-pSB1C3

1-4. 2.5 uL = 2 ng → 25 uL Mach1 → 100 uL LB-kan

Plated on kanamycin plates in 37 degree room

7/26/2016 4:36 PM

1. 15 cfu/100 uL

2. 0 cfu/100 uL

3. 15 cfu/100 uL

0 cfu/100 uL

3g. plasmid prep and restriction mapping

7 mL LB-chl

ig3f5.1A.1-2 pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

Take 3 measurements

Ig3f5.1 A1 42.22 ng/uL

Ig3f5.1 A2 52.71 ng/uL

For the restriction mapping, I cut the plasmid with PstI and EcoRI.
In theory, with PstI and EcoRI, the product of a ligation would be as follows:

Vector bp 2029bp 1392bp

Insert bp 2029bp 972bp

ig3f5.1A.1-2 bp 2029bp 2331 bp

Ig4f5.1 A1

50uL Total Volume

24uL DNA

5uL NEB 10X NEB 2.1

1uL PstI

1uL EcoRI

19uL ddH20

Ig4f5.1 A2

50uL Total Volume

19uL DNA

5uL NEB 10X NEB 2.1

1uL PstI

1uL EcoRI

24uL ddH20

Incubated overnight in 27 degree room

7/28/2016 4:04 PM

Ran gel og restriction digest

Gel Lanes:

1 2-Log DNA ladder

2 4 uL Ig3f5.1 A1

3 4 uL Ig3f5.1 A2

Both lanes 1 and 2 had bands at 2kb and one above ~2.3.

7/29/2016 11:45 AM

**Experiment iG4 - pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3 assembly**

**Purpose** to subclone the Ptac-lacOc-ampC ADC-33 insert into the pWH1266 ori-pSB1C3 to create A. baylyi-E. coli shuttle vector pWH1266 ori-Ptac-lacOc-ampC ADC-33

**Procedure**

4a. Plasmid prep

From imc229L

6 mL LB-chl

6. imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926)

6 mL LB-amp

7. BS57h4.1 tetR-PtetA-tetM-pUC57-mini (pIM1842, Mach1)

QIAcube miniprep

Eluted with 100uL EB

Gel filtration with G-50

Take 3 concentration measurement

5. imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926) 38.862ng/uL

8. imc203d6.1 Ptac-lacOc-ampC ADC-33-pMA (pIM1406, Mach1) 38.449ng/uL

4b. restriction digest to subclone Ptac-lacOc-ampC ADC-33-pMA

vector 5: desire 3404 bp, not 18 bp

40 uL imc229j3.1 pWH1266 ori-pSB1C3

5 uL 10x NEB 2.1

3 uL ddH2O

1 uL PstI

1 uL SpeI

---

50 uL

insert 8: desire 1258 bp, not 2387 bp

40 uL imc203d6.1 Ptac-lacOc-ampC ADC-33-pMA

5 uL 10x NEB 2.1

3 uL ddH2O

1 uL PstI

1 uL XbaI

---

50 uL

37 deg 7/2/2016 5:00 PM-7/3/2016 1:23 PM

Gel lanes:

1/ undigested imc229j3.1 pWH1266 ori-pSB1C3

2/ vector 5 digested

3/ undigested imc203d6.1 Ptac-lacOc-ampC ADC-33-pMA

4/ insert 8 digested

5/ 2-Log DNA Ladder

Band Isolation

vector 5(3404 bp): 0.22g

insert 8(1258 bp): 0.29g

Concentration after Gel Extraction

vector 5(3404 bp): 32.421 ng/uL

insert 8(1258 bp): 10.47 ng/uL

4c. ligation of Ptac-lacOc-ampC ADC-33 into pWH1266 ori-pSB1C3

20 fmoles pWH1266 ori-pSB1C3 vector a = 13 ng/kb x 3.4 kb = 44 ng = 1.5 uL

20 fmoles Ptac-lacOc-ampC ADC-33 b = 13 ng/kb x 1.3 kb = 17 ng = 2 uL

Enz = 1 uL 1/6 dil NEB T4 DNA ligase

Rxn vec ins enz H2O

1 1.5a 0 0 14.5

2 1.5a 0 1 13.5

3 1.5a 2b 1 11.5

+ 4 uL home made 5x Invitrogen T4 DNA ligase buffer

---

20

Put in 17 deg water bath from 7/10/2016 5:32 PM

Taken out at 7/11/2016 3:42 PM

Gel Lanes:

1 Rxn 1

2 Rxn 2

3 Rxn 3

4 2-Log DNA Ladder

4d. Heat shock transformation

**Experiment iG5 - pWH1266 ori-tetR-PtetA-tetM-pSB1C3 assembly**

**Purpose** to subclone the tetR-PtetA-tetM insert into the pWH1266 ori-pSB1C3 to create A. baylyi-E. coli shuttle vector pWH1266 ori-Ptac-lacOc-ampC ADC-33

**Procedure**

5ai. Plasmid prep

From imc229L

6 mL LB-chl

6. imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926)

6 mL LB-amp

7. BS57h4.1 tetR-PtetA-tetM-pUC57-mini (pIM1842, Mach1)

6/2/16 4:50 p.m.

Transferred 4 mls of each group of cells into each of two tubes, with repeated centrifugations and decantations. Followed the protocol outlined by the “Plasmid DNA Purification Using the QIAPrep Spin Miniprep Kit and a Microcentrifuge”, using 50 uL of EB Elution Buffer.

5aii. Gel Filtration

Using GSO Gel prepared by Cyrillus, 850ul of gel were spinned in a column at 2000rpm for 2 mins. 100ul of sample was added to the column which was placed in a new tube and centrifuged at 2000rpm for 2 mins.

5aiii. Take 3 DNA Quantification

Using the Take 3, 2 uls of each sample were placed for quantification. Results were very low, so experiment had to be repeated.

229L6: 25.239 ng/uL

229L7: 0.537 ng/uL

6/3/2016 2:15PM

5b. Plasmid prep (repeat)

From imc229L

25 mL LB-chl

6. imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926)

25 mL LB-amp

7. BS57h4.1 tetR-PtetA-tetM-pUC57-mini (pIM1842, Mach1)

37 deg rotator 7/2/16 4:54 p.m.

Transferred bacterial cells into 2.0 mL labeled microtubes with two separate transfer pipettes. Centrifuged at 13000 rpm for 1 minute. Supernatant was removed in the sinke. The procedure was performed a total of 3 times. The tubes containing only the pellets were placed in rotor adapters.

These rotor adapters were placed in the QIACube and eluted with 50uL elution buffer.

5bii. Gel Filtration

Because of the limiting capacity of the spin columns, samples were combined together in the following: 6ab, 6cd, 7ab, 7cd. Additionally, due to prior preparation of the same plasmid in two other microtubes, a gel filtration was performed with each one as well for a total of 6 separate gel filtrations.

Using GSO Gel prepared by Cyrillus, 850ul of gel were spinned in a column at 2000rpm for 2 mins. 100ul of sample was added to each column which was placed in a new tube and centrifuged at 2000rpm for 2 mins.

The final products were all pooled together as follows: 6a-d, 7a-d, and the combination of the two other microtubes prepared last week.

5biii. Take 3 DNA Quantification

Using the Take 3, 2 uls of each sample were placed for quantification.

Concentrations (ng/uL):

6. imc229j3.1 pWH1266 ori-pSB1C3 – 52.2567

7. BS57h4.1 tetR-PtetA-tetM-pUC57-mini – 22.427

Old 229G2 tet – 5.842

Because of repeatedly low obtained concentrations of 7, it may be that the plasmid does not perform well with filtration. Future experiments regarding this plasmid should take note of this fact.

7/9/2016 1:17 PM

5c. Restriction digest for insertion of BS57h4.1 tetR-PtetA-tetM-pUC57-mini into imc229j3.1 pWH1266 ori-pSB1C3

BS57h4.1 tetR-PtetA-tetM-pUC57-mini must be cut by Xba1 and Pst1 to be inserted into imc229j3.1 pWH1266 ori-pSB1C3, which is cut by Pst1 and Spe1. The following recipe outlines the amount and order of ingredients to be added to create the digest:

Restriction digest of BS57h4.1 tetR-PtetA-tetM-pUC57-mini

25 ul H2O

8 ul NeBuffer 2.1

45 ul BS57h4.1 tetR-PtetA-tetM-pUC57-mini DNA – 1 ug

1 ul Xba1

1 ul Pst1

\_\_\_\_\_\_

80 ul

Restriction digest of imc229j3.1 pWH1266 ori-pSB1C3

23 ul H2O

5 ul NeBuffer 2.1 Buffer

20 ul imc229j3.1 pWH1266 ori-pSB1C3 DNA – 1 ug

1 ul Pst1

1 ul Spe1

\_\_\_\_\_\_

50ul

Each reaction was left at 37 degrees Celsius overnight.

7/23/2016 3:00 PM

5d. Gel Filtration of tetR-PtetA-tetM-pUC57-mini and imc229j3.1 pWH1266 ori-pSB1C3

The following were combined with diluted 6X Loading Dye and run on a gel:

0.8% LE agarose in TAE buffer

1. 1 uL 5ai7/ BS57h4.1 tetR-PtetA-tetM-pUC57-mini

2. 1 uL 5c7/ BS57h4.1 tetR-PtetA-tetM-pUC57-mini

3. 1 uL 5ai6/imc229j3.1 pWH1266 ori-pSB1C3 (pIM1926)

4. 1 uL 5c6/ imc229j3.1 pWH1266 ori-pSB1C3

5. 2 uL 2-log ladder

Things and stuff and text and stuff and pictures and stuff.

#### [EXPERIMENT iG6-iG7: Subclone GusA Reporter Gene](#collapse3)

**Experiment iG6 - pWH1266 ori-Ptac-lacOc-ampC ADC-33-lacI-PT5-gusA-pSB1C3**

**Purpose** to subclone the lacI-PT5-gusA insert into the A. baylyi-E. coli shuttle vector pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3

**Procedure**

6a. plasmid prep

6 mL LB-amp

1. imc156d3.1 T1-lacI-PT5-gusA-T2-pIDTsmart (pIM556)

6 mL LB-chl

2. iG4d3.1 pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3 (pIM1932)

37 deg rotator 7/23/2016 5:11 PM

Miniprep

Gel Filtration

Concentration measurement:

Vector: pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3 (pIM1932) 62.097 ng/uL

Insert: T1-lacI-PT5-gusA-T2-pIDTsmart (pIM556) 86.667 ng/uL

6b. restriction digest to subclone T1-lacI-PT5-gusA-T2-pIDTsmart

vector: desire 4643 np, not 18 bp

25 uL iG4d3.1 pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3

8 uL 10x NEB 2.1

45 uL ddH2O

1 uL PstI

1 uL SpeI

---

80 uL

insert: desire 3696 bp, not 2123 bp

25 uL imc156d3.1 T1-lacI-PT5-gusA-T2-pIDTsmart

8 uL 10x NEB 2.1

45 uL ddH2O

1 uL PstI

1 uL XbaI

---

80 uL

37 deg 7/24/2016 12:48 PM

Gel Lanes:

1/ vector digested

2/ insert digested

3/ 2-log DNA ladder

Band Isolation

vector(4643 bp): 0.33g

insert(3696 bp): 0.31g

Concentration measurement after gel extraction

vector(4643 bp): 28.806 ng/uL

insert(3696 bp): 6.615 ng/uL

6c. ligation of T1-lacI-PT5-gusA-T2-pIDTsmart into pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3

20 fmoles pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3

vector = 13 ng/kb x 4.6 kb = 60 ng = 2.1 uL

20 fmoles T1-lacI-PT5-gusA-T2-pIDTsmart insert = 13 ng/kb x 3.7 kb = 48.1 ng = 7.3 uL

Enz = 1 uL 1/6 dil NEB T4 DNA ligase

Rxn vec ins enz H2O

1 2.1 0 0 13.9

2 2.1 0 1 12.9

3 2.1 7.3 1 5.6

+ 4 uL home made 5x Invitrogen T4 DNA ligase buffer

---

20 uL

Put in 17 deg water bath 7/25/2016 5:34 PM

Gel lane:

1/ vector original

2/ insert original

3/ vector only

4/ vector and T4 ligase

5/ vector, insert and t4 ligase

6/ (blank)

7/ 2-log DNA ladder

65 deg x 10 min

6d. transformation of E. coli Mach1 with pWH1266 ori-Ptac-lacOc-ampC ADC-33- T1-lacI-PT5-gusA-T2-pSB1C3 ligation

Heat shock transformation with E. coli

Diluted into 1/6 with H2O

1-3 = 1.25 ng ligations 1-3 = 1 uL 1/6 dil → 25 uL Mach1 → 100 uL LB-chl

Spread on plate and put in 37 deg room 7/26/2016 2:49 PM

Results: 7/27/2016 1:57 PM

1. 0 cfu/100 uL

2. 0 cfu/100 uL

3. 4 cfu/100 uL

6e. plasmid prep and restriction map

7 mL LB-chl

1-2 = iG6d3.1-2 pWH1266 ori-Ptac-lacOc-ampC ADC-33-T1-lacI-PT5-gusA-T2-pSB1C3

Plasmid prep, gel filtration

Take 3 concentrations measurement

iG6d3.1: 29.156 ng/uL

iG6d3.2: 56.917 ng/uL

Restriction mapping for

Vector: pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3

Insert: T1-lacI-PT5-gusA-T2-pIDTsmart

Product: pWH1266 ori-Ptac-lacOc-ampC ADC-33-T1-lacI-PT5-gusA-T2-pSB1C3

Digest with NheI, desired strand lengths:

Vector: 790 bp + 3871 bp

Insert: 5819 bp

Product: 790 bp + 1338 bp + 6211 bp

Digested with NheI for 30 min

Gel lanes:

1/ vector

2/ insert

3/ iG6d3.1

4/ iG6d3.2

5/ (blank)

6/ 2-Log DNA Ladder

Restriction mapping experiment repeated on 8/2/2016

Vector: pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3

Insert: T1-lacI-PT5-gusA-T2-pIDTsmart

Product: pWH1266 ori-Ptac-lacOc-ampC ADC-33-T1-lacI-PT5-gusA-T2-pSB1C3

Digest with NheI, desired strand lengths:

Vector: 790 bp + 3871 bp

Insert: 5819 bp

Product: 790 bp + 1338 bp + 6211 bp

Digest with BsmI, desired strand lengths:

Vector: 4254 bp + 407 bp

Product: 407 bp + 3047 bp + 4885 bp

5 uL mapped material

2 uL 10x NEB 2.1

12 uL ddH2O

1 uL NheI / BsmI

---

20 uL

Incubated in 37 deg 8/2/2016 3:35 PM

Gel lanes

1/ vector digested with NheI

2/ insert digested with NheI

3/ product digested with NheI

4/ vector digested with BsmI

5/ product digested with BsmI (bad loading)

6/ 2-Log DNA ladder

Three strands appeared in Lane 1 instead of two

Assume that one extra NheI site might exist

Propose to digest with XhoI and MfeI for further investigation

8/3/2016 2:48 PM

6f. transformation of A. baylyi ADP1 with ligations reactions

0.7 mL LB cultures

1. ATCC 33305

2. DSM

30 deg rotator 7/31/2016 2:32 PM

100 uL into 5 mL fresh LB (1/50)

8/1/2016 11:03 AM

A. baylyi DNA material>

1 ATCC 0.7 mL 6c1(VOO) ligation 15 uL

2 ATCC 0.7 mL 6c2(VOE) ligation 15 uL

3 ATCC 0.7 mL 6c3(VIE) ligation 15 uL

4 ATCC 0.7 mL none

5 ATCC 0.7 mL 6e2 plasmid 10 uL

6 DSM 0.7 mL 6e2 plasmid 10 uL

Incubated in 30 deg room 8/1/2016 1:20 PM

Spread onto LB ceftazidime plates and placed into 30 deg 8/1/2016 5:51 PM

0.8% LE agarose in LA buffer

1. 1 uL sample 1

2. 1 uL sample 2

3. 2 uL sample 3

4. 250 ng lambda HindIII standard

**Experiment iG7 - pWH1266 ori-Ptac-lacOc-aph(3')-VIa-lacI-PT5-gusA-pSB1C3**

**Purpose** to subclone the lacI-PT5-gusA insert into the A. baylyi-E. coli shuttle vector : pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

**Procedure**

Started 7mL cultures, possibly contaminated LB. Added kan

Vector: iG7.1 A imc229j4.1 pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3 (pIM1927)

Insert: iG7.1 B imc156d3.1 T1-lacI-PT5-gusA-T2-pIDTsmart (pIM556)

Control: iG7.1 C

Incubated in 37 degree room overnight.

Results: Growth in control so I will start more cultures with newly made LB.

Vector: iG7.1 A1 imc229j4.1 pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3 (pIM1927)

Insert: iG7.1 B1 imc156d3.1 T1-lacI-PT5-gusA-T2-pIDTsmart (pIM556)

Incubated in 37 degree room overnight.

8/10/2016 10:16 AM

Gel filtration and measurement with Take 3

iG7.1 A1 23.2 ng/uL

iG7.1 B1 59.0 ng/uL

8/11/2016 5:36 PM

7b. Restriction digest

iG7.2 A

vector: 4342 bp, not 18bp

42 uL pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

8 uL 10x NEB 2.1

28 uL ddH2O

1 uL PstI

1 uL SpeI

---

80 uL

iG7.2 B

insert: 3696 bp, not 2123 bp

17 uL imc156d3.1 T1-lacI-PT5-gusA-T2-pIDTsmart

8 uL 10x NEB 2.1

53 uL ddH2O

1 uL PstI

1 uL XbaI

---

80 uL

Incubated in 37 degree room.

8/15/2016 12:54 PM

Restriction mapping

Ran gel of uncut and cut plasmids

Lane Sample

1 DNA Ladder

2 iG7.2 A Cut pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

3 iG7.2 B Cut T1-lacI-PT5-gusA-T2-pIDTsmart

4 iG7.1 A1 Uncut pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

5 iG7.1 B1 Uncut T1-lacI-PT5-gusA-T2-pIDTsmart

8/16/2016 12:25 PM

7c. Gel extraction

Band Isolation

vector(4342 bp): 0.576 g

insert(3696 bp): 0.288 g

Concentration measurement after gel extraction

iG7.3 A vector 7.157 ng/uL

iG7.3 B insert 3.266 ng/uL

8/16/2016 7:33 PM

7d. Ligation of T1-lacI-PT5-gusA-T2-pIDTsmart into pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

20 fmoles pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

vector = 13 ng/kb x 4.3 kb = 55.9 ng = 7.8 uL

20 fmoles T1-lacI-PT5-gusA-T2-pIDTsmart

insert = 13 ng/kb x 3.7 kb = 48.1 ng = 14.7 uL

Enz = 1 uL 1/6 dil NEB T4 DNA ligase

Rxn vec ins enz H2O

1 7.8 0 0 8.2

2 7.8 0 1 7.2

3 7.8 14.7 1 0

+ 4 uL home made 5x Invitrogen T4 DNA ligase buffer (+6uL Rxn 3)

---

20 uL for Rxn 1 & 2

30uL for Rxn 3

Put in 17degree water bath

9/11/2016 3:28 PM

Ran gel to determine success of ligation

Gel 1 lane:

1/ 2-log DNA ladder

2/ vector only

3/ vector and T4 ligase

4/ vector, insert and T4 ligase

9/12/2016 12:38 PM

Gel 1 had no bands, repeated gel

Gel 2 lanes:

1/ 2-log DNA ladder

2/ cut vector

3/ cut insert

4/ vector only

5/ vector and T4 ligase

6/ vector, insert and T4 ligase

Gel 2 had no bands either. I believe that I used the wrong spin column when isolating the band in 7c. So I will redo the restriction digest with remaining plasmid from 7a.

7e. Restriction digest 2

iG7.2 A

vector: 4342 bp, not 18bp

42 uL pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3

8 uL 10x NEB 2.1

28 uL ddH2O

1 uL PstI

1 uL SpeI

---

80 uL

iG7.2 B

insert: 3696 bp, not 2123 bp

17 uL imc156d3.1 T1-lacI-PT5-gusA-T2-pIDTsmart

8 uL 10x NEB 2.1

53 uL ddH2O

1 uL PstI

1 uL XbaI

---

80 uL

Incubated in 37 degree room.

9/26/2016 3:05 PM

#### [EXPERIMENT iG8-iG9](#collapse4)

**Experiment iG8 - Repeat the experiment of iG6**

**Purpose** to prove the success of exp. iG6

**Procedure**

8a. plasmid prep

6 mL LB-amp

1. imc156d3.1 T1-lacI-PT5-gusA-T2-pIDTsmart (pIM556)

37 deg rotator 7/23/2016 5:11 PM

6 mL LB-chl

2. iG4d3.1 pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3 (pIM1932)

37 deg rotator 7/23/2016 5:11 PM

Miniprep and gel filtration

imc156d3.1 (insert): 77.698 ng/uL

iG4d3.1 (vector): 94.683 ng/uL

8b. restriction digest to subclone T1-lacI-PT5-gusA-T2-pIDTsmart

vector: desire 4643 np, not 18 bp

30 uL iG4d3.1 pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3

8 uL 10x NEB 2.1

40 uL ddH2O

1 uL PstI

1 uL SpeI

---

80 uL

insert: desire 3696 bp, not 2123 bp

30 uL imc156d3.1 T1-lacI-PT5-gusA-T2-pIDTsmart

8 uL 10x NEB 2.1

40 uL ddH2O

1 uL PstI

1 uL XbaI

---

80 uL

37 deg 8/20/2016 7:23 PM

Gel lanes:

1/ uncut vector

2/ cut vector

3/ uncut insert

4/ cut insert

5/ 2-log DNA ladder

6/ exp. iG9 A. baylyi plasmid

7/ exp. iG9 E. coli plasmid

Gel purification

Concentration:

Vector: 10.015 ng/uL

Insert: 20.215 ng/uL

8c. ligation of T1-lacI-PT5-gusA-T2-pIDTsmart into pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3

20 fmoles pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3

vector = 13 ng/kb x 4.6 kb = 60 ng = 6.0 uL

20 fmoles T1-lacI-PT5-gusA-T2-pIDTsmart insert = 13 ng/kb x 3.7 kb = 48.1 ng = 2.4 uL

Enz = 1 uL 1/6 dil NEB T4 DNA ligase

Rxn vec ins enz H2O

1 6.0 0 0 10

2 6.0 0 1 9

3 6.0 2.4 1 6.4

+ 4 uL home made 5x Invitrogen T4 DNA ligase buffer

---

20 uL

17 deg water bath 8/21/2016 4:06 PM

8d. culturing on plates

Heat shock transformation to E. coli

37 deg incubated 8/22/2016 6:11 PM

Group Content Growth

1 VOO 0 cfu

2 VOE 0 cfu

3 VIE 11 cfu

#### [EXPERIMENT iG10](#collapse5)

**Experiment iG10 - Mambalgin**

**Purpose** to Transform A. baylyi with mambalgin-1 plasmid

**Procedure**

10a. Plasmid prep

Grew LB E. coli w/ Mambalgin-1 plasmid

iG10.1 Mambalgin-1

37 deg rotator 9/23/2016 7:02 PM

Minimal growth in media. More information about the plasmid is needed before continuing. The plan is to subclone the mambalgin-1 plasmid into iG4d3.1 before transforming AB.

Plasmid prep for Mambalgin insert: 38.704 ng/uL

Plasmid prep for iG4d2 vector: 67.269 ng/uL

iG10b Restriction digest of vector and insert

Mambalgin insert:

NEB 10x 5uL

DNA sample 15uL

XbaI 1uL

PstI 1uL

H2O 28uL

-----

Total 50uL

iG4d2 vector:

NEB 10x 5uL

DNA sample 26uL

SpeI 1uL

PstI 1uL

H2O 17uL

-----

Total 50uL

iG10c Ligation

Gel filtration:

Vector: 2.67ng/uL

Insert: 2.599ng/uL

iG10d Bacteria Transformation with E.coli

Grow overnight in 37 deg

iG10d1 (VOO): 0 cfu

iG10d2 (VOE): 1 cfu

iG10d3 (VIE): 39 cfu

iG10d4 (OIE): 0 cfu

select three colonies, iG10di, iG10dii and iG10diii

iG10e Bacteria Transformation with A. baylyi

Grow overnight in 30 deg

iG10d1 (VOO): 0 cfu

iG10d2 (VOE): 0 cfu

iG10d3 (VIE): 71 cfu

iG10d4 (OIE): 0 cfu

#### [EXPERIMENT iG11](#collapse6)

**Experiment iG11 - Parts Submission Preparations**

**Purpose** Wrapping up and submiting the parts to iGEM headquarter

**Procedure**

11a. Plasmid prep

iG3(pWH1266 ori-Ptac-lacOc-aph(3')-VIa-pBS1C3): 43.974 ng/uL

iG4(pWH1266 ori-Ptac-lacOc-ampC ADC-33-pSB1C3): 37.580 ng/uL

iG6(pWH1266 ori-Ptac-lacOc-ampC ADC-33-lacI-PT5-gusA-pSB1C3): 81.72 ng/uL

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