Joe Microbe’s Self-Service Notebook

Joe Microbe


Use the Notebook Helper to format your notebook entry, then email the output with any images to rcbvz6@mst.edu!

Find the previous year’s notebook here.


29 September 2018

Lucas Dyer and Lynell Cunningham

Start: 1:00 PM

PCR of Transformation of Cry 8DA

Purpose: To prepare the transformed cells for a gel to determine the success of the digestion/ligation.

Protocol:

The Knight: Colony PCR protocol was followed. The following PCR tubes were created.

Component Tubes 1-5 Tubes 6-10 Tube 11
Taq 2X 9 μL 9 μL 9 μL
VR 0.5 μL 0.5 μL 0.5 μL
VF2 0.5 μL 0.5 μL 0.5 μL
MilliQ 10 μL 10 μL 9 μL
DNA Colony from Transfomation Plate 2% Colony from Transformation Plate 20% 1 μL L1

Thermocycler Conditions:

Temperature (°C) Time (s)
95 300
95* 30
56* 30
68* 180
68 300
4 Hold

* Denotes that this time/temperature will be cycled 30 times.

Stop: 2:30 PM

Products:

The eleven tubes from above were saved and moved to the freezer upon completion of the PCR.

Next:

A gel will be run of the PCR tubes to determine the success of the Digestion/Ligation.


28 September 2018

Lucas Dyer

Start: 1:30 PM

Transformation of Ligation of Cry 8DA

Purpose: To create cells with ligated segment of Cry DNA.

Protocol:

The Intact Genomics High Efficiency Transformation Protocol was followed to produce 54 μl of transformed cells. The transformation solution consisted of 50μL of competent cells, and 4μL of ligated DNA, taken from L1.

Stop: 3:00 PM

Results:

The transformation successfully produced live cells, further tests will determine their fidelity.

Products:

Two plates were produced: one with 20% of the solution, and one with 2%.

Plate Label
1 Transformation 20% LD 09/28/18
2 Transformation 2% LD 09/28/18

Next:

The cells will be used in a colony PCR to eventually run a gel and determine if the digestion/ligation were successful.


24 September 2018

Lucas Dyer

Start: 5:00 PM

Gel Electrophoresis of Colony PCR of Cry 8DA Transformation

Purpose: To determine the success of the transformation of the Cry 8DA sequence into competent cells and the subsequent colony PCR.

Protocol:

The Knight: Colony PCR protocol was followed for reagent volumes and thermocycler conditions. The gel was run according to standard gel procedure.

Gel Lanes

Lane Component Volume (μ)
1 Ladder 10
2 PCR 9/21 – 1 10
3 PCR 9/21 – 2 10
4 PCR 9/21 – 3 10
5 PCR 9/21 – 4 10
6 PCR 9/21 – 5 10
7 PCR 9/21 – 6 10
8 PCR 9/21 – 7 10
9 PCR 9/21 – 8 10
10 PCR 9/21 – 9 10
11 PCR 9/21 – 10 10
12 PCR 9/21 – 11 10

Stop: 6:15 PM

Results:

The gel returned large smears in each lane containing sample DNA rather than distinct bands. This confirmed the presence of DNA, but did not allow for any identification. The ladder did work indicating that the gel was run correctly and the problem instead lay in either the preparation of the samples, the PCR, or the transformation itself.

Products:

The following gel was produced.

Next:

Troubleshooting of the smear problem and repeat gels will be run using alternate methods in order to identify and isolate the desired sequence.

<a


14 September 2018

Lucas Dyer

Start: 4:00 PM

Cry Toxin Restriction Digest

Purpose: To prepare digested pieces of plasmid for use in future ligation.

Protocol:

The following digests were run in order to prepare the individual parts for the ligation of the final plasmid.

Digestion 1: Plasmid Backbone
Component Volume (μl)
O Buffer 2.5
DNA 12
EcoR1 1.0
Pst1 1.0
MilliQ Water 3.5
Digestion 2: Cry Toxin Sequence
Component Volume (μl)
Tango Buffer 2.5
DNA 6
Xba1 1.0
Pst1 1.0
MilliQ Water 9.5
Digestion 3: Promoter + RBS
Component Volume (μl)
CutSmart Buffer 2.5
DNA 2.5
Spe1 1.0
EcoR1 1.0
MilliQ Water 10

Stop: 6:00 PM

Products:

Three PCR tubes with the following contents.

Tube Label Contents
D1 Plasmid Backbone
D2 Cry Toxin Sequence
D3 Promoter + RBS

Next:

A gel will be run to determine the success of the digestions. Sequences will be isolated from the gels to be used for ligation.


27 August 2018

Ryan Baumann

Start: 12:00pm

Golden Gate Assembly of Cry8Da and Rbsc-1A into the Universal Acceptor Plasmid

Purpose: To assemble basic parts into UAP for future cloning into transcriptional units

Protocol:

Golden Gate assembly was conducted using NEB Golden Gate assembly Master Mix with BsmBI. Protocols were as follows.
27Jul18 GG1

Component Volume
UAP (5/5 MP1) 2.5 μL (100ng)
Rbsc_1A (IDT Resuspended) 6.2 uL (62 ng)
NEB T4 DNA Ligase 1 μL
NEB 10x Ligase Buffer 2 μL
BsmBI 1 μL
Sterile Water 7.3 μL
Total 20 μL

27Jul18 GG2

Component Volume
UAP (5/5 MP1) 1.8 μL (75ng)
Cry8Da (IDT Resuspended) 10 μL (100ng)
NEB T4 DNA Ligase 1 μL
NEB 10x Ligase Buffer 2 μL
BsmBI 1 μL
Sterile Water 7.3 μL
Total 20 μL

Thermocycler Conditions:
20 seconds 37°C,
(3 minutes 37°C, 4 minutes 16°C) X26 ,
5 minutes 50°C, 5 minutes 80°C,
5 minutes 16°C

Following Assembly, 2 μL of each tube was transformed following Intact Genomics ig 5-Alpha Chemically Competent Cells High Efficiency Transformation protocol. Cells were plated on Kanamycin LB Agar Plates.

Stop: 8:00 pm

Results:

Colonies were seen on all transformation plates.

Next:

Colony PCR will be conducted on plates to check for properly assembled basic parts.


 

23 August 2018 

Lynell Cunningham

Interlab Transformation Day 2

Purpose: Innoculate plates

Protocol: 

5 mL of LB Chloramphenicol were added to falcon tubes. 2 cell cultures were then added to these tubes. Tubes were put in the incubating shaker at 220 rpm.

Next: Follow the day 3 protocol for interlab.

22 August 2018

Lynell Cunningham

Interlab Transformation Day 1

Purpose: To start interlab transformations, woot woot.

Protocol: 

In wells 4B, 4D, 4F, 4H, 4J, 4L, 4N, 4P on kit plate 7, DNA was resuspended with 10uL of dH2O. 5uL of this DNA was used for the transformation according to the Intact Genomics Chemically Competent Cells pamphlet. Cells were grown on LB chloramphenical plates.

 Next: Inoculate plates in LB Chloramphenicol.

22 August 2017

Ryan Baumann, Jessica Brooks, Luke Dyer

Start: 1400

 

Colony PCR’s of previous GG assembly transformation plates to screen for proper clones

Purpose: To check if our Cry toxin, Promoter + RBS, and terminator are properly assembled into the Universal Acceptor Plasmid for assembly of transcriptional units.

Protocol:

30 Colony PCR’s were conducted using NEB’s Taq 2x Master mix protocol with the following layout.

Component Volume
Taq 2x Master Mix 10 μL
VR 0.5 μL
VF2 0.5 μL
Sterile Water 9 μL
Colony (1-30) N/A
Total Volume 20 μl

PCR 1 through 20 contain colonies from the Cry_RFC106 GG Assembly Transformation Plates

PCR 21 through 30 contain colonies from the Rbsc_1A_B0030 GG Assembly Transformation Plate

PCR 31 was conducted from a Miniprep of BBa_K1618037 labelled 9-13-17 MP3 which will be utilized as our plant terminator with the following layout.

Component Volume
NEB Taq 2X Master Mix 12.5 μL
VR 0.5 μL
VF2 0.5 μL
9-13-17 MP3 0.5 μL
Sterile Water 11 μL
20 μL

PCR 1-20 were placed in a Thermocycler with the following conditions
95°C for 30s, 30 Cycles (95°C for 30s, 54°C for 60s, and 68 °C for 120s), 68°C for 5min, and 4°C Hold.

PCR 21-31 were placed in a Thermocycler with the following conditions
95°C for 30s, 30 Cycles (95°C for 30s, 54°C for 60s, and 68 °C for 20s), 68°C for 5min, and 4°C Hold.

 

Notes:

Plates were marked with 1-30 to determine where colonies came from for future miniprep if assembled properly.

Stop: 1700

 

Results:

TBD after gels.

Next:

Gel of PCR’s 1-31 to determine if properly assembled. If properly assembled, colonies will be inoculated in liquid media for plasmid purification.


2 August 2018

Lynell, Luke, Jessica

Start: 9:40pm

 

Inoculation of transformed colonies

Purpose: Grow colonies in liquid media

Protocol:

Used LB broth with chlor

Stop:

 

Products:

Eight tubes:

RBCS-100-1
RBCS-100-2
RBCS-200-1
RBCS-200-2

From GGW

Cry-100-1
Cry-100-2
Cry-200-1
Cry-200-2

From 7/31 GG1

Notes:

Was not completed, due to flooding. Also, should have done colony PCR instead of liquid cultures.

Next:

PCR with VR and VF2, then Gel, then Miniprep, then NanoDrop


2 August 2018

Jessica

Start: 7:10pm

 

Checking Transformation Plates

Purpose: Find colonies

Protocol:

 

Notes:

CT Plate#1: No colonies
CT Plate#2: No colonies
CT Plate#3: A few colonies
CT Plate#4: A moderate amount of colonies
CT Plate#5: Tons of colonies
CT Plate#6: Even more colonies

Stop:

 

Next:

 


1 August 2018

Jessica

Start: 2:00pm

 

Blue/White Chlor Plates

Purpose: Make Blue/White Chlor X-Gal Screening Plates

Protocol:

Mix the following into 700 mL of MilliQ water:
6 g tryptone
3 g yeast extract
3 g NaCl
9 g agar

Stir solution using a magnetic stir bar and a stir plate.

Autoclave the media.

Allow flask to cool on bench or in a preheated water bath until it is approximately 50-55°C (the flask should be warm to the touch but cool enough to grasp for several seconds without burning your hands). Do not allow the solution to cool enough to solidify in the flask.

Add 725ul of 1000x Chloramphenicol 50mg/ml and 2900ul of 250x X-gal 10mg/ml to reach goal concentrations of 50mg/L and 40mg/L

Pour plates

Notes:

The amounts for tryptone, yeast extract, NaCl, and agar were changed from lab manual to align with corrected values on multiple LTP workshop sheets
2200 ul of our 250x X-gal stock and 700ul of Dr.W’s 40mg/ul were used (we ran out)
The broth spend a great deal of time in a 54-55 *C hot water bath after autoclaving and before pouring

Stop:

 

Results:

25 Blue/White Chloramphenicol Screening Plates, in large bag. Each plate is labeled with 3 orange lines (chlor), 1 yellow line (LB) and one purple line (X-gal). The sleeve is labeled 8/1/2018 LB+Chlor+X-Gal

Next:

 


1 August 2018

Jessica

Start: 12:30am

 

Redo of Arabidopsis Plates

Purpose: Previous plates were liquid, agar will be added to remedy this

Protocol:

Same as on 31 July 2018, with the addition of 7.5 g of agar. Will produce 18 plates labeled with three green lines, four of which will be used for planting (found using x/500=10.5/700)

Stop:

 

Next:

 


1 August 2018

Jessica

Start: 12:00am

 

Transformation of Cry Sequence and B0030

Purpose: Develop plates of UAP+Cry and UAP+B0030

Protocol:

Followed Intact Genomics Pamphlet

Notes: Both shaking incubators were set at 30*C, so that was the temperature during the 1 hour incubation. GG1 plates are B0030, GG- and GGW are Cry Sequence and Control (may not be in that order)

Use chlor plates

Stop:

 

Products:

Four plates, labeled:

CT1= from the GG- PCR tube, 100 ul
CT2= from the GG- PCR tube, 200 ul
CT3= from the GG W PCR tube, 100 ul
CT4= from the GG W PCR tube, 200 ul
CT5= from the 7/31 GG1 tube, 100 ul
CT6= from the 7/31 GG1 tube, 200 ul

Three tubes of cells and recovery medium:
GG- from GG- PCR
GG W from GG W PCR
GG1 from 7/31 GG1 PCR

Next:

 


31 July 2018

Jessica

Start: 9:45pm

 

Golden Gate: B0030

Purpose: To re-suspend B0030 ordered from IDT and to place it inside of a UAP

Protocol:

BsmBI Protocol

5/5 MP1 (UAP) = 2.5 ul or ~100ng
IDT B0030 = 10 ul or ~100ng
T4 Ligase = 1 ul
10x T4 Ligase Buffer = 2 ul
BsmBI = 1 ul
Water (for total of 20 ul) = 3.5 ul

Thermocycler Protocol:
20 seconds 37*C
(3 minutes 37*C, then 4 minutes 16*C) X 26 cycles
5 minutes 50*C
5 minutes 80*C
5 minutes 16*C

Notes:

B0030 was re-suspended from IDT protocol

Stop: 10:30pm

 

Results:

Re-suspension and Golden Gate Assembly

Products:

1 Tube with IDT Label of 17-Jul-2018 Rbcs-1A_B0030

1 Tube 7/31 GG 1

Next:

Transformation


31 July 2018

Jessica and Lynell

Start: 8:00pm

 

Arabidopsis Plates

Purpose: Prepare the growth medium for the development of Arabidopsis in the lab

Protocol:

 

Component Amount
Murashige and Skoog salts 2.15 g
1% Sucrose 5 ml
.05% MES .25 ml
MilliQ Water 500 ml
KOH N/A (see below)

 

Notes:

Murashige and Skoog salts were in the fridge
KOH amount is until pH is 5.7. Due to the lack of a pH meter, pH strips were used and pH was determined to be 5.5-6, so no KOH was added

Stop: 9:00

 

Results:

18 Arabidopsis Plates (18 MS Mediums)

Next:

Plant!


15 July 2018

Jessica Brooks

Start: 5:20pm

 

Gel of BBa_B0015

Purpose: Confirm quality of DNA from 24/7/17, Labeled as 7/24/17 B0015

Protocol:

From Notebook

Notes:

Used 3 uL of ethidium bromide
Used 10 uL of ladder in well closest to side of gel
Used 10 uL of sample in well closer to center of gel
Run at 130V

**Lynell ran an identical gel previous to this

Stop: 7:00

 

Results:

Ryan said lynell’s gel is good

Products:

One gel

Next:

 


5 July 2018

Jessica Brooks

Start: 12:00pm

 

PCR of BBa_B0015

Purpose: Conduct a PCR and gel of tube 24/7/17 B0015 to check DNA quality

Protocol:

New England BioLabs protocol for Taq 2x Master Mx

Used 1 ul of DNA

30 Cycles of:
95* for 20 seconds
52* for 30 seconds
68* for 30 seconds

Notes:

Used VR and VF2, which may produce an extra band at 500 and hazy ones below (shorter and dimmer than expected product)

Stop: In PCR at 12:40pm

 

Products:

One tube, labeled 7/5 PCR 1

Next:

Run gel

Should see bright band at 500 and dim hazy bands under the 129 pb B0015


2 July 2018

Jessica, Lynell, Luke

Start: 12:00

 

InterLab

Purpose: Inoculate two colonies from previous transformation

Protocol:

Followed standard protocol

Stop: 3:00pm

 

Results:

16 tubes, labeled Device #-Colony Number

Next:

 


1July 2018

Lynell Cunningham, Jessica Brooks, and Lucas Dyer

Start: 11:00am

 

Interlab Cell Measurement Transformation

Purpose: To transform kit plate Test Device DNA into competent cells for further use in the interlab.

Protocol:

Eight wells of kit plate DNA were transformed into eight samples of competent cells according to the Intact Genomics High Efficiency Protocol. Each sample was then plated at 5% and 20% onto chloramphenicol plates and grown overnight.

Stop: 1:30pm

 

Results:

The cells grew overnight on plates.

Products:

A total of sixteen plates were created

Plate Device Part Number Label
1 Negative Control BBa_R0040 Interlab Negative Control 5%
2 Negative Control BBa_R0040 Interlab Negative Control 20%
3 Positive Control BBa_I20270 Interlab Positive Control 5%
4 Positive Control BBa_I20270 Interlab Positive Control 20%
5 Test Device 1 BBa_J364000 Interlab Test Device 1: 5%
6 Test Device 1 BBa_J364000 Interlab Test Device 1: 20%
7 Test Device 2 BBa_J364001 Interlab Test Device 2: 5%
8 Test Device 2 BBa_J364001 Interlab Test Device 2: 20%
9 Test Device 3 BBa_J364002 Interlab Test Device 3: 5%
10 Test Device 3 BBa_J364002 Interlab Test Device 3: 20%
11 Test Device 4 BBa_J364003 Interlab Test Device 4: 5%
12 Test Device 4 BBa_J364003 Interlab Test Device 4: 20%
13 Test Device 5 BBa_J364004 Interlab Test Device 5: 5%
14 Test Device 5 BBa_J364004 Interlab Test Device 5: 20%
15 Test Device 6 BBa_J364005 Interlab Test Device 6: 5%
16 Test Device 6 BBa_J364005 Interlab Test Device 6: 20%

 

Next:

Cultures from plates will be inncoulated to LB+Chloramephenicol broth and grown over night.


28 June 2018

Jessica, Lynell, Luke

Start: 11:00am

 

InterLab

Purpose: Complete Calibration for Plate Reader/CFU

Protocol:

Completed Calibrations

Stop: 4:00pm

 

Next:

 


5 May 2018

Lynell Cunningham, Ryan Baumann, Lucas Dyer

Start: 1:30pm

 

Minipreps of phytobrick plasmids

Purpose: To obtain phytobrick plasmids

Protocol:

Three minipreps were performed according to the kitless miniprep procedure. Products were run through the nanodrop to determine concentration.

Stop: 4:30 pm

 

Results:

 

Sample ng/μl 260/280 260/230
5/5 MP1 39.45 1.84 2.12
5/5 MP2 34.38 1.92 2.01
5/5 MP3 37.94 1.76 1.45

 

Products:

 

Label Source Description
5/5 MP1 5/2 BC3 UAP/BBa_P10500 in pSB1C3
5/5 MP2 5/2 BC4 Alpha 1/BBa_P10501 in pSB1K3
5/5 MP3 5/2 BC5 Alpha 2/BBa_P10503 in pSB1K3

 

Next:

Digest each miniprep with EcoRI and PstI run products on gel to verify identity


3 May 2018

Jessica Brooks, Lynell Cunningham, and Kent Gorday

Start: 16:15

 

Minipreps of amilCP and phytobrick plasmids

Purpose: To obtain amilCP in pSB1C3, along with the five (UAP + alpha and omega) phytobrick plasmids

Protocol:

Seven minipreps were performed according to the kitless miniprep procedure. Products were nanodropped to determine concentration.

Stop: 19:00

 

Results:

 

Sample ng/μL 260/280 260/230
5/3 MP1 172.31 1.87 1.88
5/3 MP2 123.26 1.84 2.25
5/3 MP3 50.63 1.78 2.04
5/3 MP4 41.19 1.68 1.54
5/3 MP5 30.47 1.72 1.71
5/3 MP6 8.30 1.26 0.99
5/3 MP7 1.65 0.87 0.78

 

Products:

 

Label Source Description
5/3 MP1 5/2 BC1 amilCP in pSB1C3 (higher concentration?)
5/3 MP2 5/2 BC2 amilCP in pSB1C3 (cleaner prep?)
5/3 MP3 5/2 BC3 UAP/BBa_P10500 in pSB1C3
5/3 MP4 5/2 BC4 Alpha 1/BBa_P10501 in pSB1K3
5/3 MP5 5/2 BC5 Alpha 2/BBa_P10503 in pSB1K3
5/3 MP6 5/2 BC6 Omega 1/BBa_P10505 in pSB1K3
5/3 MP7 5/2 BC7 Omega 2/BBa_P10507 in pSB1K3

 

Next:

Digest each miniprep with EcoRI and PstI run products on gel to verify identity


2 May 2018

Ryan Baumann and Lucas Dyer

Start: 4:00

 

Broth cultures of cloning vectors

Purpose: To obtain good preps of several cloning vectors

Protocol:

One chloramphenicol broth culture and four kanamycin broth cultures were inoculated

Stop: 4:30

 

Products:

The following broth cultures were created

Label Source Description
5/2 UAP BC3 5/1 Transformation UAP/BBa_P10500 in pSB1C3
5/2 Alpha 1 BC4 5/1 Transformation Alpha 1/BBa_P10501 in pSB1K3
5/2 Alpha 2 BC5 5/1 Transformation Alpha 2/BBa_P10503 in pSB1K3
5/2 Omega 1 BC6 5/1 Transformation Omega 1/BBa_P10505 in pSB1K3
5/2 Omega 2 BC7 5/1 Transformation Omega 2/BBa_P10507 in pSB1K3

 

Next:

Miniprep all broth cultures


2 May 2018

Kent Gorday

Start: 15:00

 

Broth cultures of amilCP in pSB1C3

Purpose: To obtain good preps of amilCP in pSB1C3

Protocol:

Two LB + chloramphenicol broth cultures of amilCP in pSB1C3 were inoculated

Stop: 15:20

 

Products:

 

Label Source Description
5/2 BC1 5/1 amilCP transformation amilCP in pSB1C3
5/2 BC2 5/1 amilCP transformation amilCP in pSB1C3

 

Next:

Miniprep amilCP in pSB1C3 from broth cultures


1 May 2018

Luke Dyer and Ryan Baumann

Start: 4:30

 

Transformations of 2018 Cloning vectors

Purpose: Transformation of cloning vectors and parts needed for summer research.

Protocol:

Transformation of Golden braid destination vectors, UAP, and AmilCP were conducted following Intact genomics 5 Alpha electro competent cells protocol.

UAP and AmilCP were plated on Chloramphenicol plates while GB Destination Vectors were plated on Kanamycin Plates

Stop: 7:30

 

Products:

 

Part Transformed
Alpha 1/BBa_P10501
Alpha 2/BBa_P10503
Omega 1/BBa_P10505
Omega 2/BBa_P10507
UAP/BBa_P10500
AmilCP/BBa_K592009

 

Next:

Grow Overnight and inoculate cultures in liquid media.


2-5-18

Ryan Baumann

Start: 10:00AM

 

DNA Purification of AmilCP and BBa_J04500

Purpose: To purify the promoter and chromoproterin for future miniprojects.

Protocol:

Plasmid minipreps were conducted following Missouri S&T iGEM’s Lab manual 2.5 kitless miniprep protocol. DNA was resuspended in 40 μL TE buffer and concentration was tested with a Thermo Scientific Nanodrop 1000.

Nanodrop Results:

Name DNA Conc. 260/280
1-22-18 MP1 (amilCP) 121.73 1.89
1-22-18 MP2 (amilCP) 158.92 1.89
2-1-18 MP1 (BBa_J04500) 119.82 1.87
2-1-18 MP2 (BBa_J04500) 127.71 1.90

 

Stop: 10:15 AM

 

Products:

 

Label Source Description
2-1-18 MP1 BBa_j04500 from 2014 kit plate 3 BBa_J04500 inducible promoter
2-1-18 MP2 BBa_j04500 from 2014 kit plate 3 BBa_J04500 inducible promoter
1-22-18 MP1 amilCP Chromoprotein from 2016 kit plate amilCP blue Chromoprotein in pSB1C3
1-22-18 MP2 amilCP Chromoprotein from 2016 kit plate amilCP blue Chromoprotein in pSB1C3

 

Next:

 


30 October 2017

Ryan Baumann

Start: 5:00PM

 

Cloning efficiency testing of new Universal Acceptor Plasmid

Purpose: To test the cloning efficiency of the Universal Acceptor Plasmid after deletion of the unexpected 10 bp region via site directed mutagenesis PCR

Protocol:

Golden gate assemblies were performed to insert TNTR3 gblock into our universal acceptor plasmid.
Various molar ratios of TNTR3: Universal acceptor plasmid were tested.
Molar ratios of 1:2, 1:1, 2:1 (recommended ratio), 4:1, and 8:1 were tested for the new plasmid. A 2:1 ratio assembly was performed using our original UAP. A negative control assembly was performed with no inserts added.

Golden gate assemblies contained the following:
1:2 Ratio

Component Volume
TNTR3 (17.64 ng)
New UAP (100ng) 1 μL
10X Ligase Buffer 2 μL
Ligase 1 μL
BsmBI 1 μL
MilliQ H2O up to 20μL

1:1 ratio

Component Volume
TNTR3 (35.28 ng)
New UAP (100ng) 1 μL
10X Ligase Buffer 2 μL
Ligase 1 μL
BsmBI 1 μL
MilliQ H2O up to 20μL

2:1 Ratio

Component Volume
TNTR3 (70.56 ng)
New UAP (100ng) 1 μL
10X Ligase Buffer 2 μL
Ligase 1 μL
BsmBI 1 μL
MilliQ H2O up to 20μL

4:1 Ratio

Component Volume
TNTR3 (141.12 ng)
New UAP (100ng) 1 μL
10X Ligase Buffer 2 μL
Ligase 1 μL
BsmBI 1 μL
MilliQ H2O up to 20μL

8:1 Ratio

Component Volume
TNTR3 (282.24 ng)
New UAP (100ng) 1 μL
10X Ligase Buffer 2 μL
Ligase 1 μL
BsmBI 1 μL
MilliQ H2O up to 20μL

2:1 Ratio with original UAP

Component Volume
TNTR3 (70.56 ng)
Original UAP (100ng) 1 μL
10X Ligase Buffer 2 μL
Ligase 1 μL
BsmBI 1 μL
MilliQ H2O up to 20μL

Negative Control

Component Volume
TNTR3 None
UAP None
10X Ligase Buffer 2 μL
Ligase 1 μL
BsmBI 1 μL
MilliQ H2O up to 20μL

All samples were run with the following thermocycler conditions:
– 20 seconds @ 37°C,
– (3 minutes @ 37°C, 4 minutes @ 16°C) X26
– 5 minutes @ 50°C
– 5 minutes @ 80°C
– 5 minutes @ 16°C

Following assembly, 1 uL of each sample was transformed on to Chloramphenicol Blue-White Screening plates following Intact Genomics ig 5-Alpha chemically competent cell High efficiency Transformation protocol. Dillutions of 20% and 5% were plated and spread using sterile glass beads. A positive control transformation was performed using 1 uL of original UAP.

Colonies were incubated at 37C for 30 hours.

Blue and white colonies were counted on all plates.

Stop: 1:45AM

 

Results:

See project page

Next:

Boston!


25 October 2017

Ryan Baumann

Start: 3

 

Blunt end Ligation of 10/22/17 PCR1 and transformation

Purpose: Ligation of UAP back to itself after removal of 10bp sequence.

Protocol:

Blunt end ligation was performed uisng NEB T4 DNA ligase protocol. 2 uL of 10/22/17 OJM PCR 1 was ligated in 20 uL reaction.

Reaction was conducted at room temperature for 2 hours.

After Ligation, 2 uL of 10/25/17 L1 was transformed using Intact Genomics ig 5-Alpha chemically competent cell High efficiency Transformation protocol. 50% and 5% dilutions were plated onto Chloramphenicol plate.

Notes:

Check colony growth

Stop: 5:00

 

Products:

 

Label Source Description
10/25/17 L1 10/22/17 PCR1 UAP Ligated back to itself following removal of 10bp fragment

 

Next:

 


23 October 2017

Ryan Baumann

Start: 8:45PM

 

Colony PCR’s of 10/18/2017 Transformations of PhoB and TNTr3, gels of Golden Gate Round two PCR’s and 10/23/17 PCR 1 and 2

Purpose: To check assembly of golden gate round two assemblies and test OJM1 and OJM2 primers.

Protocol:

18 Colony PCRs were conducted using Taq 2x Master Mix protocol. 9 Colonies from PhoB and TNTR3 transformation plates were used as template.

PCR 1-9 – TNTR3
PCR 10-18 PhoR

Three 1% agarose gels were prepared with 2 uL of ethidium bromide
Gel 1:

Lane(s) Component
1 2 Log purple DNA ladder
2 TNTR3 PCR1
3 2 Log purple DNA ladder
4-10 TNTR3 PCR 2-3 and 5-9
11 2 Log purple DNA ladder
12-20 PhoR PCR 1-9

Gel 2:

Lane(s) Component
1 2 Log purple DNA ladder
2-8 10/23/17 cPCR’s 1-7 (Q92X31)
13 2 Log purple DNA ladder
14-20 10/23/17 cPCR’s 8-14 (PhoB)
23 10/23/17 OJM PCR1
24 10/23/17 OJM PCR 2

Gel 3:

Lane(s) Component
1 2 Log Purple DNA Ladder
2-8 10/23/17 cPCR 15-21 (O22527)
11 2 Log Purple DNA Ladder
12-18 10/23/17 cPCR 22-28 (Q8LDU4)

 

Notes:

Insert Gel Pictures

Stop: 10:30

 

Next:

 


23 October 2017

Jeremy Mesa

Start: 5:00 pm

 

Purpose: To perform a PCR with mutagenesis primers in order to delete a short base pair sequence from plasmid.

Protocol:

Contents of PCR tubes:

Q5 oJM1 oJM2 UAP Milli-Q
PCR 1 12.5 μL 1.25 μL 1.25 μL 1 μL 4 μL
PCR 2 12.5 μL 1.25 μL 1.25 μL 0 μL 5 μL

PCR 2 was a negative control.

Thermal Cycler Program:

Temp (°C) Time Cycles
Step 1 98 30 sec 0
Step 2 98 5 sec 24
Step 3 65 20 sec 24
Step 4 72 75 sec 24
Step 5 72 2 min 0
Step 6 4 Infinite

 

Stop: 5:45

 

Next:

 


23 October 2017

Ryan Baumann, Jessica Brooks, Tiffany Kuhnert, Lucas Dyer

Start: 4:15pm

 

Colony PCR of 10/23/2017 Q92x31 5%, Q22527 5% , PhoB 50% , and Q8LD44 5% , golden gate round 2 transformation plates

Purpose: To confirm assembly of title parts with their promoters and terminators

Protocol:

28 Colony PCRs were conducted using Taq 2x Master Mix protocol. 7 colonies from each plate were run along with a RFP Construct postitive control and milliQ water negative control.

1-7 Q92X31
8-14 PhoB
15-21 O22527
22-28 Q8LDU4

Reaction Set Up:

Component Volume (μ)
Taq 2x Master Mix 7.5
VR 0.3
VF2 0.3
MilliQ 6.9
Colony1-34

Thermocycler Conditions:

Temp. (C) Time Repeats
Step 1 95 5 min 0
Step 2 95 30 sec 35
Step 3 51 30 sec 35
Step 4 68 3 min 35
Step 5 68 5 min 0
Step 6 4

 

Stop:

 

Next:

Run Gels of all the colony PCRs


18 October 2017

Ryan Baumann

Start: 2:30PM

 

Plasmid mini preps of Q9LX31, Q8LDU4, and O22527 transformations. Golden gate assemblies of PhoB, PhoR, TNTR3, Q9LX31, Q8LDU4, and O22527 and transformation on to Kanamysin Blue white screening plates

Purpose: Purify degreening plasmids and assemble signal transduction pathway, periplasmic binding protein, and degreening parts into transcriptional units.

Protocol:

Minipreps were conducted using iGEM lab manual 2.5 kitless miniprep protocol. Samples were resuspended in 50 uL of TE buffer.

DNA Concentration were calculated using Thermoscientic Nanodrop 1000.
Nanodrop Results:

Sample DNA Conc. (ng/μL) 260/280
10/17 MP3 (O22527) 122.1 2.11
10/17 MP4 (O22527) 364.9 1.86
10/17 MP5 (Q9LX31) 326.9 1.88
10/17 MP6 (Q9LX31) 280.2 1.91
10/18 MP1 (Q8LDU4) 76.7 1.93
10/18 MP2 (Q8LDU4) 59.3 1.90
10/18 MP3 (K1467101) 47.4 1.93
10/18 MP4 (K1467101) 100.3 1.93

Golden Gate Assemblies of PhoB (9/8/17 MP3), PhoR (9/13/17 MP1), TNTR3 (9/8/17 MP1), O22527 (10/17/17 MP4), Q9LX31 (10/17/17 MP5), Q8LDU4 (10/18/17 MP2) were performed following the NEB Golden Gate assembly kit protocol into golden braid alpha 1 acceptor plasmid (BBa_P10501).

The plant pho promoter (9/8/17 MP2) was added to the degreening parts.

BBa_K1467101 added to TNTR3, PhoR, and PhoB.

BBa_K1618037 (9/13/17 MP3) was added to all parts.

2 uL of each golden gate assemblies were transformed using Intact Genomics ig 5-Alpha chemically competent cell protocol. 50% and 5% dilutions were plated onto Kanamysin Blue white screening plates.

 

Stop: 8:30PM

 

Results:

Check for colonies

Products:

 

Label Source Description
10/17 MP3 O22527 O22527 in the UAP
10/17 MP4 O22527 O22527 in the UAP
10/17 MP5 Q9LX31 Q9LX31 in the UAP
10/17 MP6 Q9LX31 Q9LX31 in the UAP
10/18 MP1 Q8LDU4 Q8LDU4 in the UAP
10/18 MP2 Q8LDU4 Q8LDU4 in the UAP
10/18 MP3 K1467101 K1467101 in the UAP
10/18 MP4 K1467101 K1467101 in the UAP
10/18 GG1 9/8/17 MP3 PhoB with BBa_K1467101 Promoter and BBa_K1618037 Terminator
10/18 GG2 9/13/17 MP1 PhoR with BBa_K1467101 Promoter and BBa_K1618037 Terminator
10/18 GG3 9/8/17 MP1 TNTR3 with BBa_K1467101 Promoter and BBa_K1618037 Terminator
10/18 GG4 10/17 MP4 O22527 with Plant Pho Promoter and BBa_K1618037 Terminator
10/18 GG5 10/17 MP5 Q9LX31 with Plant Pho Promoter and BBa_K1618037 Terminator
10/18 GG6 10/18/17 MP2 Q8LDU4 with Plant Pho Promoter and BBa_K1618037 Terminator

 

Next:

 


17 Oct. 2017

Jeremy Mesa

Start: 2:30

 

Purpose: To run PCR with mutagenesis primers and a gel following to visualize the results of the reaction.

Protocol:

PCR:
Contents of the tube, 20 μL total

Tube Q5 oJM1 oJM2 UAP Milli-Q
PCR 1 10 μL 1 μL 1 μL 1 μL 7 μL

Thermal Cycler Program:

Temp. (°C) Time Repeats
Step 1 98 30 sec 0
Step 2 98 10 sec 30
Step 3 65 20 sec 30
Step 4 72 2 min 30
Step 5 72 2 min 0
Step 6 4 Infinite 0

1% Agarose gel was prepared with 3 μL Ethidium Bromide.

Lane 1 Ladder 20 μL
Lane 4 PCR 1 15 μL

 

Notes:

Program name on thermal cycler is saved as PCR10-6.

Stop: 4:00

 

Results:

PCR lane of gel was found to have 4 bands at about 2500bp, 1500bp, 600bp, and less than 100bp. The lane was streaked behind the largest base pair band.

Next:

 


10 15 2017

Lynell Cunningham, Luke Dyer, Tiffany Kuhnert

Start: 3:00

 

Colony PCR and Gel of 9/30/17 Golden gate assemblies of degreening parts

Purpose: To confirm assembly of Q9LX31, Q8LDU4, and O22527 into the universal acceptor plasmid.

 

Protocol:

17 Colony Pcr’s were conducted using Taq 2x Master mix protocol. 5 Colonies from each 9/30/17 golden gate reaction were run along with a RFP Construct postitive control and milliQ water negative control.

Reaction setup:

Component Volume
Taq 2X Master Mix 10 μL
VR .4 uL
VF2 .4 uL
MilliQ 9.2 uL
Colony 1-15

Thermocycler Conditions:
95C for 5 minutes, 35 Cycles of: (95C for 20 seconds, 66C for 30 sec, 68C for 1min), 68C for 5 minutes, and 4C hold

All samples were run on a 1% agarose gel with the following setup:
Lane 1 and 11: 2 log purple DNA ladder
Lane 2-6 are Colonies 1-5 from Q8LDU4
Lanes 7,8,9,10,and 12 are Colonies 6-10 from O22527
Lanes 13-17 are Colonies 11-15 for Q9LX31
Lane 18 is a water negative control
Lane 19 is the RFP Construct positive control

After imaging, one colony from each golden gate transformation was grown in broth culture containing Chloramphenicol

Notes:

Add Gel Picture

Stop: 10:00pm

 

Next:

Grow successfully assembled cultures, miniprep, and round two assemblies


10 October 2017

Ryan Baumann

Start: 4:30PM

 

Gel of 9 October 2017 Golden Gate and Colony PCR’s

Purpose: To confirm proper assembly of previous golden gate assemblies

Protocol:

A 1% agarose gel with 1.5 μL Ethidium Bromide and 2 Log Purple DNA Ladder.10 μL of samples were loaded into wells.

Well Sample
1 2 Log purple DNA Ladder
2 1
3 2
4 3
5 4
6 5
7 6
8 7
9 8
10 9
11 2 Log Purple DNA Ladder
12 10
13 11
14 12
15 13
16 14
17 15
18 16
19 17
20 18
21 10/9/17 A1
22 10/9/17 A2
23 10/9/7 A3

 

Notes:

Insert Gel Pic

Stop: 6:00:00PM

 

Results:

Analyze Gel Results

Next:

 


30 September 2017

Kent Gorday, Lynell Cunningham, Lucas Dyer, Jeremy Mesa

Start: 16:35

 

Diagnostic PCRs of round 2 assemblies and assembly of degreening gblocks into UAP

Purpose: To check addition of constitutive promoter and terminator to coding sequences, and assemble degreening coding sequences into Universal Acceptor Plasmid for submission.

Protocol:

Six PCRs were performed from round 2 assemblies:

9/30 A1-6
10 μL 2X Q5 Master Mix
7 μL MilliQ Water
1 μL Template Plasmid
1 μL VF2
1 μL VR
20 μL Total Volume
Temperature (°C) Time (sec)
98 30
98c 8
65c 22
72c 93
72 120
4

cycle X32

Three Golden Gate reactions into the Universal Acceptor Plasmid were performed:

9/30 GG1-3
4.9 μL 100 ng/μL Q9LX31 gblock
2.5 μL UAP
2 μL 10X T4 DNA Ligase Buffer
1 μL T4 DNA Ligase
1 μL BsmBI
8.6 μL MilliQ Water
15.1 μL Total Volume
5.9 μL 100 ng/μL O22527 gblock
2.5 μL UAP
2 μL 10X T4 DNA Ligase Buffer
1 μL T4 DNA Ligase
1 μL BsmBI
7.6 μL MilliQ Water
14.1 μL Total Volume
5.8 μL 100 ng/μL Q8LDU4 gblock
2.5 μL UAP
2 μL 10X T4 DNA Ligase Buffer
1 μL T4 DNA Ligase
1 μL BsmBI
7.7 μL MilliQ Water
14.2 μL Total Volume
Temperature (°C) Time (sec)
37 20
37c 180
16c 240
50 300
80 300
16 300

cycle X32

A 1.2% agarose gel was run with PCR products:

Well No.
3 Ladder
4 A1
5 Ladder
6 A2; Bad Load
7 A4
8 A3; Bad Load
9 A5
10 A6

 

Stop: 20:40

 

Results:

ADD GEL PIC

Products:

 

Label Source Description
9/30 A1 9/18 MP1 + pro/ter amplified
9/30 A2 9/18 MP2 + pro/ter amplified
9/30 A3 9/18 MP3 + pro/ter amplified
9/30 A4 9/18 MP4 + pro/ter amplified
9/30 A5 9/18 MP5 + pro/ter amplified
9/30 A6 9/18 MP6 + pro/ter amplified
9/30 GG1 Q9LX31 gblock GUN4 CDS assembled into Universal Acceptor Plasmid
9/30 GG2 O22527 gblock Chlorophyllase-1 CDS assembled into Universal Acceptor Plasmid
9/30 GG3 Q8LDU4 gblock RCCR CDS assembled into Universal Acceptor Plasmid

 

Next:

Colony PCR and patch to a new plate a few white colonies from each round 2 assembly transformation.


9-21-17

Ryan Baumann

Start: 1:30PM

 

PCR and Gel of 18 September 2017 minipreps (again)

Purpose: To identify if phoR, phoB, and TNTR3are properly assembled in transcriptional units with BBa_K1618037 and BBa_K1467101

Protocol:

Six 20 μL PCR’s of 9/18/17 MP 1-6 were run using containing: Q5 HIgh Fidelity 2X Master Mix, 0.4 μL VR and VF2 (10 μM solutions), The following template concentrations, and MilliQ Water
up to 20 μL.

Template Volume
9/18/17 MP1 1 μL
9/18/17 MP2 1 μL
9/18/17 MP3 1 μL
9/18/17 MP4 1 μL
9/18/17 MP5 4 μL
9/18/17 MP6 5 μL

A positive control was run using a 1μL of RFP construct in psb1C3.
A negative control was run using MilliQ water.

Samples were run in the thermocycler in the following conditions:

98C for 30 seconds
32 cycles of: 98C for 8 seconds, 65C for 22 seconds, and 72C for 85 seconds
72C for 120 seconds
4C Hold

All PCR’s were run on a 1% agarose gel containing 1.5 μL of ethidium bromide

Well Sample
1 2 Log Purple DNA Ladder
3 PCR1(MP1)
4 PCR2(MP2)
5 PCR3(MP3)
6 PCR4(MP4)
7 PCR5(MP5)
8 PCR6(MP6)
9 Positive Control
10 Negative Control

 

Notes:

Add Gel Picture

Stop:

 

Results:

Continue to troubleshoot PCR conditions. No conclusive results from gel.

Next:

Redo PCR’s again


21 September 2017

Ryan Baumann

Start: 2:00 PM

 

PCR and Gel of 18 September 2017 minipreps

Purpose: To identify if phoR, phoB, and TNTR3are properly assembled in transcriptional units with BBa_K1618037 and BBa_K1467101

Protocol:

Six 20 μL PCR’s of 9/18/17 MP 1-6 were run using containing:
300ng of template DNA
Q5 High Fidelity 2X Master Mix,
0.2 μM VR and VF2
and water up to 20 μL

A positive control was run using an RFP construct in psb1C3.
A negative control was run using MilliQ water.

Samples were run in the thermocycler in the following conditions:

98C for 30 seconds
32 cycles of: 98C for 8 seconds, 65C for 22 seconds, and 72C for 85 seconds
72C for 120 seconds
4C Hold

All PCR’s were run on a 1% agarose gel containing 1.5 μL of ethidium bromide

Well Sample
1 2 Log Purple DNA Ladder
3 PCR1(MP1)
4 PCR2(MP2)
5 PCR3(MP3)
6 PCR4(MP4)
7 PCR5(MP5)
8 PCR6(MP6)
9 Positive Control
10 Negative Control

 

Notes:

Insert Gel Picture

Stop: 5:30PM

 

Next:

TBD


14 September 2017

Ryan Baumann

Start: 2:00PM

 

PCR and Gel of 13 September 2017 minipreps

Purpose: To identify if Trg_PhoR and BBa_K1618037 Terminator are properly assembled in the UAP

Protocol:

PCR’s of 9/13/17 MP1 (9/14/17 PCR1), MP2 (9/14/17 PCR2), and MP3 (9/14/17 PCR3), and an RFP positive control (9/14/17 PCR4) were run with the following conditions:

Component Volume
Taq 2X Master Mix 10 μL
VR (10μM) 0.4 μL
VF2 (10μL) 0.4 μL
Samples 0.7 μL
MilliQ 8.5 μL
Total 20 μL

PCR’s were run using taq2kbp program following taq 2x master mix protocol.

Annealing temperatures calculated using IDT’s annealing tempature protocoL

All Colony PCR’s were run on a 1% agarose gel containing 2.0 μL ethidium bromide.

Gel Layout:

Lane Component
1 2 Log Purple DNA Ladder
3 9/14/17 PCR1
5 9/14/17 PCR2
7 9/14/17 PCR3
9 9/14/17 PCR4

 

Notes:

Insert Gel Picture

Stop: 6:00PM

 

Next:

Round two of golden gate assemblies.


6 September 2017

Ryan Baumann

Start: 5:00PM

 

Golden Gate Assembly of BBa_K1618037 Terminator into UAP and Transformations

Purpose: To assemble plant terminator into the universale acceptor plasmid using BsmBI Type IIS endonuclease and transform on to chloramphenicol blue white screening plates.

To transform amilCP chromoprotein (BBa_K592009) and eforRed Chromoprotein (BBa_K592012) on to chloramphenicol plates.

Protocol:

Reaction setup:

UAP 0.20 μL (~60 ng)
BBa_K1618037 5 μL (~50 ng)
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1 μL
BsmBI 1 μL
MilliQ Water 10.8 μL
20 μL

Samples Ran:
– 20 seconds @ 37°C,
– (3 minutes @ 37°C, 4 minutes @ 16°C) X26
– 5 minutes @ 50°C
– 5 minutes @ 80°C
– 5 minutes @ 16°C

2 μL of the Terminator assembly, 1 μL of AmilCP, and 1 μL of eforRed were transformed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol.

The 1% and 10% dilutions were plated for each. AmilCP and eforRed were transformed on to chloramphenicol plates. The terminator assembly was plated on Chlor blue-white screening plates.

Stop: 10:30pm

 

Products:

 

Label Source Description
9/6/17 AmilCP 2017 Kit Plate 1 Well 19E amilCP resuspended from kit plate
9/6/17 eforRed 2017 Kit Plate 7 Well 15I eforRed resuspended from kit plate

 

Next:

Check for colony growth. Plasmid mini prep desired colonies.


2 September 2017

Ryan Baumann

Start: Colony PCR and Gel of 8/31/17 Transformants

 

Confirm colonies with properly assembled plant parts with promoter and terminator

Purpose: To find properly assembled plasmids of PhoB, PhoR, and TNTr3 with promoter and terminator within alpha 1 acceptor plasmid.

Protocol:

20uL Colony PCR’s were run for 7 PhoB colonies, 7 TNTr3 colonies, and 6 PhoR colonies.

Sample colony PCR

Component Volume
Taq 2x Master Mix 10 μL
VR 0.8 μL
VF2 0.8 μL
MilliQ 8.4 μL
Colonies 1-20

PCR’s were run using taq2kbp program following taq 2x master mix protocol with annealing temperatures calculated using IDT’s annealing tempature protocol.

All Colony PCR’s were run on a 1% agarose gel containing 1.5 μL ethidium bromide.

Gel Layout

Lane(s) Sample
1 2 Log Purple Ladder
3-9 PhoB colonies 1-7
10-12 TNTr3 COlonies 1-3
13 2 Log Purple Ladder
15-18 TNTR3 4-7
19-24 PhoR colonies 1-6

 

Notes:

Insert Gel pic

Stop:

 

Next:

Troubleshoot gel results.


1 September 2017

Benjamin Bleitz

Start: 1:00 pm

 

Preparation of Arabidopsis growth medium

Purpose: to prepare a proper growth medium for Arabidopsis in a laboratory setting

Protocol:

 

Component Amount
Murashige and Skoog salts 2.15 g
1% Sucrose 5 ml
0.05% MES 0.25 ml
MilliQ H2O total of 500 ml
KOH Until pH adjusted to 5.7

Sucrose, MES and final solution all Autoclaved for 30 minutes on liquid cycle
Plates poured

Stop: 3:30

 

Results:

18 plates prepared for plant growth

Products:

18 MS Mediums

Next:

Test growth mediums


31 August 2017

Ryan Baumann

Start: 3:00 PM

 

Addition of Promoter and Terminator to PhoR, PhoB, and TNTR3

Purpose: Addition of BBa_K1467101 Promoter and BBa_K1618037 Terminator to PhoR, PhoB, and TNTR3 into BBa_P10501 Alpha 1 acceptor plasmid via golden gate assembly using NEB golden gate assembly kit.

Protocol:

20 uL Reactions were set up following NEB golden gate assembly mix using 75ng of BBa_P10501 as the destination plasmid and 100ng of each insert.

All samples were run at 37C for 1 hour followed by 55C for 5 minutes.

1 uL of each assembly were transformed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol.

20% and 2% dilutions were then plated on Kanamysin selective blue white screening plates.

Stop: 7:00PM

 

Results:

Growth of white colonies was seen from assembly transformation.

Next:

Colony PCR from transformation plates to confirm proper assembly


23 August 2017

Ryan Baumann

Start: 3:00 PM

 

PCR of August 18th plasmid minipreps with VR and VF2 and gel extraction

Purpose: To verify correct assembly of fragments in the Universal Acceptor Plasmid and extract correct bands from gel

Protocol:

~100 ng of purified template DNA was amplified following the iGEM Lab Manual 2.5 PCR Protocol using Taq 2x MM, VR, VF2, and MilliQ H2O.

A positive control was run using linearized Psb1T3 backbone.

A negative control as run with a water template.

Sample Reaction:

Sample Volume
Taq 2X Master Mix 12.5 μL
VR 1 μL
VF2 1 uL
Plant Pho (100ng/1uL) 1 uL
Water 9.5 μL
Total 25 μL

Samples were run in thermocycler using Taq 2X master mix recommend protocol with appropriate annealing temperature for VR and VF2.

All PCR’s were run in a 1% agarose gel containing 2 μL ethidium bromide.

Gel Layout:

Lane Sample
1 2 Log Purple DNA ladder
3 Negative Control
4 TNTR3
5 Plant Pho
6 Positive Control
7 PhoB
8 PhoR

Bands containing Assembled TNTR3 (~1100BP), Plant Pho (~450BP), PhoB (~1100BP), and PhoR (~1800 BP) were cut from the gel and DNA was purified using Amicon Ultra-DA Kit for DNA extraction from agarose gel.

Notes:

Add Gel Picture

Stop: 5:06 PM

 

Products:

 

Label Source Description
8/23/17 Plant Pho 8/18/17 plant Pho Miniprep Extracted plant pho DNA from gel
8/23/17 Pho R 8/18/17 PhoR miniprep extracted PhoR from gel
8/23/17 Pho B 8/18/17 PhoB miniprep Extracted PhoB from Gel
8/23/17 TNTR3 8/18/17 tntr3 miniprep Extracted TNTr3 from gel

 

Next:

 


18 August 2017

Ryan Baumann

Start: 11:00am

 

Plasmid Miniprep of colonies from 8/9/17 COlony PCR’s

Purpose: To isolate plasmids from colonies with correctly assembled PhoB, OhoR, Plant pho, and TNTR3

Protocol:

Colonies that showed positive bands from PCR were innoculated into broth cultures with chloramphenicol and grown up overnight.

Plasmid DNA was purified using the MST iGEM Lab Manual 2.5 Kitless miniprep protocol and suspended in 40uL of TE buffer.

2uL of sample of was run on Thermoscientific Nanodrop 1000.

Nanodrop results:

Sample Concentration (ng/uL) 260nm/280nm
PhoB 263.2 1.84
PhoR 98.6 1.80
Plant Pho 99.8 1.86
TNTr3 236.0 1.82

 

Stop: 1:40PM

 

Products:

 

Label Source Description
8/18/17 MP1 PhoB Colonies from 6/26/17 Transformation PhoB in the UAP
8/18/17 MP2 PhoR from 6/26/17 Transformation PhoR in the UAP
8/18/17 MP3 Plant Pho from 6/26/17 Transformation Plant Pho in the UAP
8/18/17 MP4 TNTR3 from 6/26/17 Transformation TNTr3 in the UAP

 

Next:

PCR with VR and VF2 and extract band containing correctly assembled plasmid for sequencing. If correctly assembled, perform golden gate assembly to add Promoter and terminator to each plant part.


9 August 2017 to 10 August 2017

Ben Bleitz

Start: 2:00 PM of 9 August 2017

 

PCR and Gels of 18 Colonies from the PhoR Plate

Purpose: Correctly identify assembled of plasmid containing PhoR in universal acceptor

Protocol:

PCRs were conducted for marked colonies 1-18 on PhoR

Component Volume
Taq 2x Master Mix 10μl
VR 0.8μl
VF2 0.8μl
MilliQ 8.4μl
Colonies 1-18

Samples were run through the Taq2bkp program and then frozen for the next day
All Samples were run on a 1% agarose gel with .75 uL Ethidium Bromide with 2 Log Purple DNA ladder, the following day.

Stop: 1:00 PM of 10 August 2017

 

Results:

Gel pics or it didn’t happen

Next:

Inoculate culture from appropriate culture


8 August 2017

Ryan Baumann

Start: 4:00PM

 

Colony PCR of 6/26/17 TNTR3 transformation

Purpose: To identify correctly assembled plasmid containing TNTr3 in the universal acceptor plasmid

20 μL Sample reaction

Component volume
Taq 2X Master Mix 10 μL
VR 0.8 μL
VF2 0.8 μL
MilliQ 8.4 μL
Colonies 1-16

A positive Control was conducted and run

Component volume
Taq 2X Master Mix 10 μL
VR 0.8 μL
VF2 0.8 μL
MilliQ 7.4 μL
7/24/17 K608002 1 uL

A negative control PCR was conducted and run

Component volume
Taq 2X Master Mix 10 μL
VR 0.8 μL
VF2 0.8 μL
MilliQ 8.4 μL
20 μL

All Samples were run on a 1% agarose gel with .75 uL Ethidium Bromide wit 2 Log Purple DNA ladder.

Protocol:

PCR’s of 16 colonies were run with VR and VF2 primers, Taq 2x Master mix, and MilliQ Water

 

Notes:

Add gel Picture

Stop: 9:00 PM

 

Results:

TBD

Next:

Examine Gel and find if colonies match correctly assembled plasmid


3 August 2017

Ben Bleitz

Start: 2:00

 

Running Gel of Plant Pho, PhoB, PhoR, TNTR3

Purpose: Running Gels of repeat PCR

Protocol:

 

Lane Component
1 10 uL NEB 2 Log Purple DNA ladder
3 10 uL 7/27/17 PCR 5
5 10 uL 7/27/17 PCR 1
6 10 uL 7/27/17 PCR 2
7 10 uL 7/27/17 PCR 3
8
9 10 uL 7/24/17 K608002 (+ Control)
10 10 uL MilliQ Water (- Control)

 

Stop: 5:00

 

Results:

Somehow managed to repeat mistakes from last week. Will repeat, for a third and hopefully final time for last week.

Next:

DONT MESS UP


2 August 2017

Ben Bleitz

Start: 1:00 pm

 

PCR of 7/25 L1, 7/24 Plant Pho and 6/29 MP1 (PhoB), MP2 (PhoR), MP6 (TNTR3)

Purpose: Repeat of previous PCR (and eventual gel) from 27 June 2017. Gel scheduled to be completed following day (3 August 2017)

Protocol:

Plant Pho PCR:

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
7/24/17 Plant Pho 3 μL
MilliQ 7.5 μL
Total 25 μL

Pho B Pcr:

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
6/29/17 MP1 1.5 μL
MilliQ 9 μL
Total 25 μL

TNTR3 Pcr:

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
6/29/17 MP6 2.0 μL
MilliQ 8.5 μL
Total 25 μL

PhoR Pcr:

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
6/29/17 MP2 1.5 μL
MilliQ 9 μL
Total 25 μL

7/25/17 L1

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
7/25/17 L1 5 μL
MilliQ 5.5 μL
Total 25 μL

 

Notes:

PCR was completed. Gel will be ran following day

Stop: 4:20 pm (#Blazeit)

 

Results:

PCRs frozen after thermocycler completed. Will be determined upon gel electrophoresis completion on the following day.

Products:

 

Label Source Description
PCR 1 7/24/17 Plant Pho 7/24/17 Plant Pho amplified with VR and VF2
PCR 2 6/29/17 MPI 6/29/17 MPI amplified with VR and VF2
PCR3 6/29/17 MP6 6/29/17 amplified with VR and VF2
PCR4 6/29/17 MP2 6/29/17 amplified with VR and VF2
PCR5 7/25/17 L1 7/25/17 L1 amplified with VR and VF2

 

Next:

Run gel


27 June 2017

Erin Nischwitz, Ben Bleitz

Start: 11:00 am

 

PCR and gels of 7/25 L1, 7/24 Plant Pho and 6/29 MP1 (PhoB), MP2 (PhoR), MP6 (TNTR3)

Purpose: To verify assembly of these constructs

Protocol:

Five PCR’s were ran with VR and VF2 using Q5 2x Master mix Protocol

Plant Pho PCR:

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
7/24/17 Plant Pho 3 μL
MilliQ 7.5 μL
Total 25 μL

Pho B Pcr:

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
6/29/17 MP1 1.5 μL
MilliQ 9 μL
Total 25 μL

TNTR3 Pcr:

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
6/29/17 MP6 2.0 μL
MilliQ 8.5 μL
Total 25 μL

PhoR Pcr:

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
6/29/17 MP2 1.5 μL
MilliQ 9 μL
Total 25 μL

7/25/17 L1

Component Volume
Q5 2x Master Mix 12.5 μL
VR 1μL
VF2 1 μL
7/25/17 L1 5 μL
MilliQ 5.5 μL
Total 25 μL

A 1% agarose gel was ran containing all five PCRs, a positive control, and a Negative control

Lane Component
1 10 uL NEB 2 Log Purple DNA ladder
3 10 uL 7/27/17 PCR 5
5 10 uL 7/27/17 PCR 1
6 10 uL 7/27/17 PCR 2
7 10 uL 7/27/17 PCR 3
8 10 uL 7/27/17 PCR 4
9 10 uL 7/24/17 K608002 (+ Control)
10 10 uL MilliQ Water (- Control)

 

Notes:

– Add Gel Picture

-Accidentally used circular DNA as a positive control, so do not expect a valid + Control

Stop: 3:30

 

Results:

TBD

Products:

 

Label Source Description
PCR 1 7/24/17 Plant Pho 7/24/17 Plant Pho amplified with VR and VF2
PCR 2 6/29/17 MPI 6/29/17 MPI amplified with VR and VF2
PCR3 6/29/17 MP6 6/29/17 amplified with VR and VF2
PCR4 6/29/17 MP2 6/29/17 amplified with VR and VF2
PCR5 7/25/17 L1 7/25/17 L1 amplified with VR and VF2

 

Next:

Trouble Shoot


24 July 2017

Ryan Baumann

Start: 11:00 AM

 

Plasmid Miniprep of 7/21/17 Transformations

Purpose: Prepare plasmids for future use

Protocol:

Kitless miniprep protocol from igem lab manual version 2.5
Plasmids were suspended in 35 μl of 1x TE buffer

Stop: 12:40PM

 

Results:

Samples tested on Thermoscientific Nanodrop 1000

Label DNA Conc. 260/280
BBa_B0015 893.5 ng/uL 1.95
BBa_K608002 573.5 ng/uL 1.88
Plant Pho 165.7 ng/uL 1.93

Skype Backpack

Products:

 

Label Source Description
7/24/17 B0015 2017 Kit Plate 3 well 3F B0015 Terminator in psb1c3
7/24/17 Plant Pho Plant Pho top and bottom strand Plant pho in UA plasmid
7/24/17 K608002 2017 Kit Plate 1 well 3O K608002 Promoter and RBS in psb1c3

 

Next:

 


25/June/2017

Ben Bleitz

Start: 1:30

 

Digestion and Ligation of BBa_K608002 and TNTR3_E.Coli

Purpose: To prepare DNA Samples for Transformations

Protocol:

Two double digestions were performed
BBa_K608002 Double digestion

Component Volume
ECORI Enzyme 1 μl
SpeI Enzyme 1 μl
K608002 Insert 3.5 μl
Tango Buffer 2.5 μl
MilliQ H2O 17 μl

TNT_E.Coli Double Digestion

Component Volume
ECORI Enzyme 1 μl
Xbal Enzyme 1 μl
TNTR3_E.Coli Vector 5 μl
Tango Buffer 2.5 μl
MilliQ H2O 15.5 μl

Products were 25/7/2017 d1 and 25/7/2017 d2
Ligation was then performed

Component Volume
25/7/2017 d1 (insert) 1.5 μl
25/7/2017 d2 (vector) 2 μl
T4 Ligase Buffer 2 μl
T4 Ligase 1 μl
13.5 MilliQ H2O 13.5

 

Stop: 5:30

 

Products:

 

Label Source Description
25/7/2017 d1 BBa_K608002 K608002 cut with ECORI & SPeI
25/7/2017 d2 TNTR3_E.Coli TNTR3_E.Coli cut with ECORI & Xbal
25/7/2017 L1 d1 and d2 DNA formed from d1 insert and d2 vector

 

Next:

PCR with VR and VF 2 to confirm products


20/7/2017

Ben Bleitz

Start: 2:30

 

Bacterial promoter/ terminator and plant pho Transformations

Purpose: Transform components for later prep and kit plate

Protocol:

Transformations were performed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol.

Transformant DNA Volume Plate Dilutions
Plant Pho 1μl 2 and 20%
BBa_B0015 1μl 2 and 20%
BBa_K608002 1μl 2 and 20%

 

Stop: 5:30

 

Results:

All samples in incubation. Results will be noted at the end of 24 hour incubation period

Products:

 

Label Source Description
BBa_B0015 2017 Kit Plate 3 well 3F Transformed on chlorophenicol plate
BBa_K608002 2017 Kit Plate 1 well 3O Transformed on chlorophenicol plate
Plant Pho Plant Pho top and bottom strand Transformed on Blue/White chlorophenicol plate

 

Next:

Inoculate in broth culture with appropriate antibiotic for future miniprep.


10 July 2017

Ryan Baumann

Start: 12:30

 

Golden Gate assembly of Plant Pho Top and Bottom strand into UA plasmid

Purpose: To assemble PlantPho Oligos into UAP to prepare for assembly of Transcriptional Unit

Protocol:

 

UA MP1 (100ng) 0.3 μL
PlantPho Top Strand (100 ng) 1 μL
PlantPho Bottom Strand (100 ng) 1 μL
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1 μL
BsmBI 1 μL
MilliQ H2O 13.7 μL
20 μL

Ran at:
– 20 seconds @ 37°C,
– (3 minutes @ 37°C, 4 minutes @ 16°C) X26
– 5 minutes @ 50°C
– 5 minutes @ 80°C
– 5 minutes @ 16°C

Stop: 5:00

 

Products:

 

Label Source Description
7/10/17 Plant Pho Top & Bottom Strand Oligos Plant Pho Oligos cut with BsmBI and Ligated into UAP

 

Next:

Transform on to Chlor Blue white Screening Plates


29 June 2017

Ryan Baumann, Ben Bleitz

Start: 1:30

 

Miniprep of 6/26/17 transformations

Purpose: Prepare plasmids for future use

Protocol:

Kitless miniprep protocol from igem lab manual version 2.5
Plasmids were suspended in 35 μl of 1x TE buffer

Stop: 3:30

 

Results:

Samples tested on Thermoscientific Nanodrop 1000

Label DNA Concentration (ng/μl) 260/280
MP1 412.7 1.84
MP2 399.6 1.83
MP3 290.2 1.82
MP4 722.4 1.80
MP5 406.4 1.81
MP6 307.4 1.84

 

Products:

 

Label Source Description
6/29/17 MP1 PhoB-VP64 Transformation plate PhoB-VP64 transformed into UA Plasmid
6/29/17 MP2 Trg-PhoR Transformation plate Trg-PhoR transformed into UA Plasmid
6/29/17 MP3 Golden Braid Omega 1 Plasmid Transformation plate Golden Braid Omega 1 plasmid
6/29/17 MP4 Golden Braid Omega 2 Plasmid Transformation plate Golden Braid Omega 2 plasmid
6/29/17 MP5 TNTR3 E. Coli Transformation plate TNTR3 E. Coli in PSB1C3 backbone
6/29/17 MP6 TNTR3 Transformation plate TNTR3 transformed into UA plasmid

 

Next:

TBD


6/26/2017

Ryan Baumann, Ben Bleitz, Erin Nischwitz

Start: 5:00 pm

 

Chemical Transformations

Purpose: To transform PhoB-VP64, Trg_PhoR, Plant Pho, and TNTR3 on to chloramphenicol blue white screening plates.

To transform TNTR3_E Coli (6/22/17 L1) on to chloramphenicol plate

To transform Golden Braid Omega 1 and Omega =1 plasmids on to Streptomycin plates.

 

Protocol:

Transformations were performed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol.

Transformant DNA volume Plate Dillutions
TNTR3 1 μL 2% and 20%
Trg_PhoR 1 μL 2% and 20%
PhoB_VP64 1 μL 2% and 20%
Plant Pho 2 μL 2% and 20%
6/22/17 L1 (TNTR3 E coli) 3 μL 5% and 50%
Omega 1 1 μL 2% and 20%
Omega Two 1 μL 2% and 20%

 

Stop: 7:45

 

Next:

Check for colonies and inoculate cultures


6/26/2017

Golden Gate Assembly into Universal Acceptor Plasmid

Start: 12:00pm

 

Assembly of PhoB_VP64, Trg_PhoR, TNTR3, and Plant Pho into BBa_P10500 plasmid.

Purpose: To assemble gblocks into UAP to prepare for assembly of Transcriptional Unit

Protocol:

Trg_PhoR-Gblock:

UA MP1 (100ng) 0.30 μL
Trg_PhoR (100ng) 1 μL
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1 μL
BsmBI 1 μL
MilliQ H2O 14.7 μL
20 μL

PhoB_VP64-gblock

UA MP1 (100ng) 0.30 μL
PhoB_VP64 (100ng) 1 μL
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1 μL
BsmBI 1 μL
MilliQ H2O 14.7 μL
20 μL

TNT_R3-gblock

UA MP1 (100ng) 0.30 μL
TNT_R3 (100ng) 1 μL
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1 μL
BsmBI 1 μL
MilliQ H2O 14.7 μL
20 μL

PlantPho Oligos Top and Bottom Strands

UA MP1 (100ng) 0.30 μL
PlantPho Top Strand (100 ug) 1μL
PlantPho Bottom Strand (100 ug) 1μL
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1μL
BsmBI 1μL
MilliQ H2O 13.7 μL
20 μL

All Samples Ran:

– 20 seconds @ 37°C,
– (3 minutes @ 37°C, 4 minutes @ 16°C) X26
– 5 minutes @ 50°C
– 5 minutes @ 80°C
– 5 minutes @ 16°C

 

Notes:

100 μg of top and bottom strand oligos were accidentally used instead of 100ng.

Stop: 4:45 PM

 

Products:

 

Label Source Description
6/26 TNTR3 TNTR3-GBlock TNTR3-GBlock cut with BsmBI and Ligated into UAP
6/26 Plant Pho Plant Pho Top & Bottom Strand Oligos Plant Pho Oligos cut with BsmBI and Ligated into UAP
6/26 PhoR Trg_PhoR-Gblock Trg_PhoR-Gblock cut with BsmBI and Ligated into UAP
6/26 PhoB PhoB_VP64 Gblock PhoB_VP64 Gblock cut with BsmBI and Ligated into UAP

 

Next:

Transform assembled plasmids on to Blue White Screening Plates with appropriate antibiotic resistance


6/26/2017

Golden Gate Assembly into Universal Acceptor Plasmid

Start: 12:00pm

 

Assembly of PhoB_VP64, Trg_PhoR, TNTR3, and Plant Pho into BBa_P10500 plasmid.

Purpose: To assemble gblocks into UAP to prepare for assembly of Transcriptional Unit

Protocol:

Trg_PhoR-Gblock:

UA MP1 (100ng) 0.30 μL
Trg_PhoR (100ng) 1 μL
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1 μL
BsmBI 1 μL
MilliQ H2O 14.7 μL
20 μL

PhoB_VP64-gblock

UA MP1 (100ng) 0.30 μL
PhoB_VP64 (100ng) 1 μL
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1 μL
BsmBI 1 μL
MilliQ H2O 14.7 μL
20 μL

TNT_R3-gblock

UA MP1 (100ng) 0.30 μL
TNT_R3 (100ng) 1 μL
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1 μL
BsmBI 1 μL
MilliQ H2O 14.7 μL
20 μL

PlantPho Oligos Top and Bottom Strands

UA MP1 (100ng) 0.30 μL
PlantPho Top Strand (100ng) 1μL
PlantPho Bottom Strand (100ng) 1μL
10X T4 DNA Ligase Buffer 2 μL
T4 DNA Ligase 1μL
BsmBI 1μL
MilliQ H2O 13.7 μL
20 μL

All Samples Ran:

– 20 seconds @ 37°C,
– (3 minutes @ 37°C, 4 minutes @ 16°C) X26
– 5 minutes @ 50°C
– 5 minutes @ 80°C
– 5 minutes @ 16°C

 

Stop: 4:45 PM

 

Products:

 

Label Source Description
6/26 TNTR3 TNTR3-GBlock TNTR3-GBlock cut with BsmBI and Ligated into UAP
6/26 Plant Pho Plant Pho Top & Bottom Strand Oligos Plant Pho Oligos cut with BsmBI and Ligated into UAP
6/26 PhoR Trg_PhoR-Gblock Trg_PhoR-Gblock cut with BsmBI and Ligated into UAP
6/26 PhoB PhoB_VP64 Gblock PhoB_VP64 Gblock cut with BsmBI and Ligated into UAP

 

Next:

Transform assembled plasmids on to Blue White Screening Plates with appropriate antibiotic resistance


23/6/17

Ben Bleitz

Start: 2:50pm

 

Ligation of PsbIC3 (Vector) and 6/21/17 DI (insert)

Purpose: To create recombinant DNA for transformation

Protocol:

 

T4 Ligase Buffer 2μL
PsbIC3 (Vector) 4μL
6/21/17 DI (Insert) 3μL
MilliQ H2O 10μL
T4 DNA Ligase 1μL

Thermal Cycler
16°C for 30 minutes
80°C for 20 minutes

Stop: 4:00pm

 

Results:

6/23/17 L1

Next:

E. Coli Transformation


6/21/2017

Ben Bleitz

Start: 2:00

 

Double Digestion of TNT R3_E.Coli

Purpose: To prepare TNT R3_E.Coli for submission into the registry

Protocol:

 

Volume Component
2.5 μL 10X Tango Buffer
1 μL ECORI
1 μL PSTI
15 μL TNTR3_E.Coli (10 ng/μL)
5.5 μL MilliQ H2O
25 μL in Total

Ran @ 37° C for 1 hour
Heat kill @ 80°C for 30 minutes

Stop: 4:00 PM

 

Products:

 

Label Source Description
6/21/17 D1 TNTR3_E.Coli TNTR3_E.Coli cut with ECORI and PSTI

 

Next:

Ligate into PsbIC3