Team:Missouri Rolla/Description

From Idea to Project

We were inspired to join the fight against Emerald Ash Borers (EABs) - an invasive beetle to the Ash tree - because their infestation is a local problem in Missouri. We were familiar with the use of Bacillus Thuringiensis (BT) toxins in agricultural crops, and wanted to explore using BT toxins to help create Ash trees resistant to EABs.

Though it is unknown exactly how long Emerald Ash Borers have been in the US, they were first identified in Michigan in 2002. Since then, they have been identified in 30 U.S. states and have caused millions of dollars in damage. EABs are a beetle which originated in Asia; it is expected that these beetles came to the U.S. on infested wood materials. Because Asian ash trees coevolved with the beetle, they are immune, but American ash trees do not have this same benefit. Adult beetles lay their larva in the bark, where they begin to develop. As they grow, they feed on the tree and eat through the phloem. After generations, this is enough to kill the trees as nutrients can no longer travel into the canopy.

Ash trees are incredibly common in the U.S. Following the devastation caused by the Dutch Elm disease, urban foresters settled on ash trees to replace the fallen elms, because they had and have a great balance of quality attributes: fast growing, no smelly fruits, large canopies, and beautiful fall colors. This makes the disease especially devastating to urban areas, where ash trees can make up upwards of even 90% of trees in a city. Current methods for prevention of EABs can be too expensive for prophylaxis for many cities and neighbors. Additionally, often the beatles have caused too much damage by the time they are noticed for treatment to be effective as the trees are too damaged.

Emerald Ash Borer spread as of August 2017. Photo credit; Emerald Ash Borer Information Network

After focusing on EABs, we looked into viable options for genetically engineering resistance into trees. This may not protect trees that are already planted, but when speaking with urban foresters, we spoke about the importance of tree diversity. If every city were to have more varied tree species, a problem EABs or Dutch Elm could not be as devastating again. By leaving ash trees on the market, this would encourage urban foresters to keep this diversity and prevent this same problem from happening again and again. Though multiple methods seemed appropriate, we began investigating Bacillus thuringiensis (Bt) and, based on talks with Monsanto scientists, this would be especially appropriate if used alongside an RNAi mechanism. Bt is a rod shaped, Gram-positive, spore-forming bacterium. Bt strains produces crystal proteins, called δ-endotoxins or pore-forming toxins. These crystal proteins often show insecticidal abilities to specific species within the insect orders Lepidoptera, Diptera, and Coleoptera. After ingestion, Cry proteins are solubilized in the midgut and are processed into active toxins by gut proteases. Activated Cry toxins bind specific receptors on the midgut epithelial cell brush border membrane. While searching for a Cry toxin that may be effective against EABs, we came across Dr. Leah Bauer and her team at US Department of Forestry Service and the University of Michigan. They showed that Adult Emerald Ash Borers could be impacted by a Bt SDS-502, which expresses Cry8Da. After ingestion of a formulation of Bt SDS-502 sprayed on leaves, 89% mortality was seen within 96 hours of feeding. Cry8Da has been currently approved for the treatment of multiple scarab beetles, the Japanese beetle, and multiple wood boring members of the genus Agrilus.

Once we identified Cry8Da, we explored methods to improve the specificity of this toxin for Emerald Ash Borers. From this, we discovered iGEM Wageningen 2016’s project who worked to engineer a Cry3a protein to kill a common honey bee pest - the mite varroa destructor. To prove the protein could potentially combat the pest, the team conducted a fluorophore release assay. This assay was used to determine how well the mutated Cry3a protein could lyse open Brush Border Membrane Vesicles by measuring released fluorescence levels from the vesicles.

Using these ideas, our group hoped to engineer the Cry8Da toxin to increase specificity towards adult Emerald Ash Borers which would be expressed within leaf tissues of Arabadopsis thaliana to target feeding adult EABs. To begin, we had the Cry8Da gene synthesized (UniProtKB - D5H3I7) from IDT after being codon optimized to remove internal restriction sites. Along with this, we had the Arabidopsis rbcS-1A promoter synthesized to be our model of tissue specific gene expression. We planned to utilize primer directed mutagenesis of suspected binding sites of Cry8Da for our targeting. The Cry8Da protein consists of three domains. Research suggests domain II alpha loop structures are responsible for host receptor binding in coleopteran insects. Further research has indicated that alpha loop 2 in domain II is likely associated with toxicity in adult beetles. Our group planned to target this region for mutagenesis. In the end, we were unable to assemble these parts into a functioning transcriptional unit following Golden Braid (GB) Assembly standard using iGEM supplied GB.2.0 plasmid backbones. Due to these issues, we were unable to conduct our mutagenesis experiments on our Cry8Da gene to attempt to screen for appropriate toxins. Our group was thankful to receive killed EAB’s from Robert C. Venette at the USDA Northern Research Station, but, due to handling issues in transit, the samples obtained were not able to be utilized for assembly of our BBMVs for future testing.

After meeting with Bayer scientists and discussing our project, our team has developed directions to advance our project in the future. We have connections to receive new EABs for assembly of BBMV for our testing. We are looking into RNAi gene silencing as a method of targeting EABs. Along with this, we are exploring the possibility of isolating BT spores from EABs to search for possible natural EAB specific Cry toxins. Lastly, our group plans to continue efforts to improve our gene construction ability to further develop our current method of treatment.

See attributions for references used and experts spoken to regarding the above information.

Members of iGEM Missouri Rolla plant arabidopsis seedlings

Notebook

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.

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

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.

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

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.

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.

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.

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:

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

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:

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

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

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)

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

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

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

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%

28 June 2018

Jessica, Lynell, Luke

Start: 11:00am

InterLab

Purpose: Complete Calibration for Plate Reader/CFU

Protocol:

Completed Calibrations

Stop: 4:00pm

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

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

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

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

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

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