Notebook

7/1, MRH: A plasmid prep was done on all of the cultures following the protocol found in the qiagen spin miniprep kit (used the altered protocol written by Jake and Barney).

7/6, MRH: The plasmid prep samples were nanodropped with the following concentrations:

• B0015: 286.7 ng/ul
• BCD12: 215.6 ng/ul
• DVK: 270.0 ng/ul
• J23100: 62.9 ng/ul

7/12, DTJ: The genes from IDT were used along with parts B0015, BCD12, DVK, and J23100 in a golden gate reaction using restriction enzyme BSA1. Of the 10 plasmids created, 5 plasmids have constitutive promoters and 5 have inducible promoters.

7/19, DTJ: A liquid culture of DH5a E. coli were made competent, flash frozen in liquid nitrogen, and stored at -80 C.

7/20, DTJ: Genes from IDT along with parts B0015, BCD12, DVK, and J23100 were used to create 2 of the 5 plasmids with constitutive promoters and 2 of the 5 plasmids with inducible promoters. Once representative plasmids from each group are confirmed working, the rest will be created. The resulting plasmids are stored at -20 C overnight for the transformation the next day. Reaction tubes are labeled 21, 23, 26, and 28 recipes for which are stored in an Excel file.

7/21, TRP: Chemical transformation with GG reactions 21, 23, 26, 28, and DVK (as a positive control) were completed using competent E. coli transformation protocol. 3uL of DNA was added to 30uL of DH5α that were made on 7/19. 967uL of SOC was added to the mixture instead of 950uL.

7/26, DTJ: As previous transformation/culture attempts were unsuccessful, the transformation protocol was repeated using the 3uL of the DVK plasmid, 30uL DH5α competent cells created in the lab, and 30uL purchased DH5α competent cells as a positive control. The sample with purchased DH5α cells is labeled “+ ctrl” and the sample with cells created in the lab is labeled “iGEM made”

7/27, DTJ & CLO: As the transformation and culture of DVK worked properly in both purchased cells and the competent cells created in our lab, an additional 10 transformations were performed. Golden gate constructed plasmids 21, 23, 26, and 28 and DVK were transformed into both purchased cells and our competent cells. Samples labeled with “+” indicate that they are made with purchased cells. As there was not a sufficient amount of competent cells to be used for all the transformations, DVK+ was made with purchased competent cells from the previous day that was stored at 4 C. 28 and DVK were also made with our competent cells from the previous day that were stored at 4 C. The rest of the samples were made with cells that were stored at -80 C.

After shaking at 37°C for 3 hours samples 21, 23, 26, 28, DVK, 21+, 23+, 26+, 28+, and DVK+ were plated. Samples 26, 28, 26+, and 28+ fell off the shaker, so growth was expected to be less for those samples. Each sample was plated at 10^0, 10^-1, and 10^-2 (Dilution protocol). They were put in the incubator at 37°C.

7/28, CLO: Plates were taken out of the incubator in the AM and put in the fridge. All samples grew except for sample 28.

7/29, MRH and CLO: Made media and poured plates. Streaks were made of 4 isolated colonies for 21, 23, 26, and 28+ (28 did not grow).

8/01, CLO: Streaks were in the incubator over the weekend so they were overgrown and thrown away. Single colonies were taken from 21, 23, 26, and 28+ and streaked onto new plates to grow at 37°C overnight.

8/02, JDS & CLO: Plate 23 did not grow and only 1 of the 4 streaks on plate 26 grew. Because of this, we used the original transformation plates of 23+ and 26+ for the liquid cultures/isolation streaks. The original plates for 23 and 26 had little growth. The isolation streaks of 28+ and 21 grew and were used for their liquid cultures. 4 different colonies from each strain (21, 23+, 26+, 28+) were inoculated in liquid culture, then streaked on new plates using (2 colonies per plate). We ended the day with 16 liquid cultures and 8 plates.

8/03, DTJ & JDS & CLO: DTJ: Plasmid prepped of all 16 cultures from 8/02 and eluted with 50ul of water. JDS: Using Geneious software, the combination of a Cla1 and EcoR1 enzyme was found to digest pGEC 021-030 with a unique fragment pattern on a digital electrophoresis gel. CLO: The plasmid prep samples were nanodropped with the following concentrations:

• 21-1: 313.4 ng/ul
• 23+-2: 156.0 ng/ul
• 26+-3: 41.8 ng/ul
• 28+-4: 246.2 ng/ul

All 16 plasmid preps were digested with both the EcoR1 and Cla1 enzymes in a single pot reaction. Each tube contained 2.5ul rCutSmart Buffer, 0.5ul EcoR1, 0.5ul Cla1, and 21.5ul of the respective plasmid prep. The 16 tubes were put into the thermocycler and incubated at 37°C for 40 minutes then 65°C for 20 minutes.

8/04, TRP & DTJ: The gel was run following the 1.5% Gel protocol (Larger gel). The samples were loaded in numerical order as listed on 8/2 and below. When imaging, we initially saw no bands at all. Barney suggested soaking the gel in a Midori Green solution before imaging again. After letting it sit for 30ish minutes, we imaged again and saw the bands below. One thing to note is the top left corner is a bit wavy. The gel looked completely set when pulling the comb out but was not in that area. Barney said it would be okay to run and would turn out still.

• Wells 1, 10, and 19: 1kb Ladder
• Well 2: 21-1
• Well 3: 21-2
• Well 4: 21-3
• Well 5: 21-4
• Well 6: 23+-1
• Well 7: 23+-2
• Well 8: 23+-3
• Well 9: 23+-4
• Well 11: 26+-1
• Well 12: 26+-2
• Well 13: 26+-3
• Well 14: 26+-4
• Well 15: 28+-1
• Well 16: 28+-2
• Well 17: 28+-3
• Well 18: 28+-4

8/09, MRH & JDS: Poured ~15 LBA+Amp plates and struck 2 strains from the -80C freezer.

8/10, TRP: Made liquid cultures of RBS-HE and mScarlet with a negative control using LB media. Grown overnight in 37C shaker.

8/11, CLO: Plasmid prepped RBS-HE and mScarlet. Concentrations: RBS-HE= 140.1 ng/ul, mScarlet= 198.8 ng/ul. A restriction digest of the plasmid preps was done with Bsa1.

8/12, CLO: A small 1.5% gel was made (60 mL 0.5x TAE, 0.9G agarose, 5 uL midori green).

• Well 1: 1kb ladder
• Well 2: mScarlet (C99)
• Well 3: RBS-HE (BCD2)


8/15, JDS & CLO: Diluted DNA parts to standard concentrations for Golden Gate cloning. All parts will now be at the correct concentration to add 1.0ul to Golden Gate cloning reactions.

Parts	Current conc. (ng/ul)	Target conc. (ng/ul)	DNA (ul)	H2O (ul)	Total (ul)
(1)	100	26	13.0	37.0	50
(2)	100	26	13.0	37.0	50
(3)	100	26	13.0	37.0	50
(4)	100	26	13.0	37.0	50
(5)	100	19.5	9.8	40.2	50
(6)	100	33.8	11	22	33
J23100_AB	62.9	55.9	*	*	20
BCD2_BC	140.1	57.2	40.8	59.2	100
B0015_DE	286.7	57.2	20.0	80.0	100
DVK_AE	270	70.2	20.0	57.0	77
mScarlet-I	198.8	72.8	36.6	63.4	100

Used the “GG Cloning Calculator” Excel sheet to determine the amount of each component for each Golden Gate reaction.

Thermocycler settings:
30x cycles:

• 37°C for 1 minute
• 16°C for 1 minute

1x cycle:

• 60°C for 5 minutes

4C hold

8/16, DTJ & MRH & CLO: SOC media was made by autoclaving 400mL SOB media, adding 8mL autoclaved 1M MgSO4, and adding 8mL 1M glucose. Transformations were done for samples 25, 31, 35, 36, 37, and DVK_AE (plasmid prep).

pGEC021 was struck from wedge 1 of the 8/1 plate, and pGEC023+ was struck from colony 3 on the 7/26 transformation plate.25, 31, 35, 36, 37, and a DVK control were also plated. All plates were incubated at 37°C overnight.

8/17, CLO: 25, 31, 35, and 37 had very little growth, so they were left in the incubator for an additional day. Liquid cultures of 21, 23, and 36 were made– two of each, one had arabinose added at the start, the other will have arabinose added tomorrow.

8/18, CLO & MRH: All liquid cultures grew, and 36+ arabinose was the only pink one. Made more LBB.

8/22, JDS: Designed new CD sequences (11) through (21). All sequences were designed with Bsa1 sites flanking the CD region. Then flanked by Bpi1 sites for the option to clone into the pOGG006 vector for immortalization in a plasmid form.Then an additional AAA at the 5’ end and a TTT at the 3’ end. (21) has the AmilCP gene which is another chromoprotein (blue/purple) from the 2013 iGEM team. TwistBioscience website identified hard to synthesize regions in the GS linker and changed the codons to make synthesis possible. The sequences are as follows:

• (11)His-meffRed-Linker-CBDfimi
• (12)His-meffRed-Linker-CBDclos
• (13)CBDfimi-Linker-meffRed-His
• (14)CBDclos-Linker-meffRed-His
• (15)meffRed-His_NegControl
• (16)His-mScarlet-Linker-CBDfimi
• (17)His-mScarlet-Linker-CBDclos
• (18)CBDfimi-Linker-mScarlet-His
• (19)CBDclos-Linker-mScarlet-His
• (20)mScarlet-His_NegControl
• (21)AmilCP-His_NegControl

Additionally, pGEC036 and pGEC037 were restruck from their 8/16 transformation plate. Three colonies from each plate were struck onto a new LBA+Km plate divided into 3 wedges.

8/23, DTJ & CLO & JDS: Blunt ligation and transformation. Parts used in the blunt ligation were 1, 5, and +( the positive control in the kit) and tubes were labeled as such.

Diluted 21-3, 23-3, 26-2, 28-1 to 30 ng/uL (were 414.8 ng/ul, 258.5 ng/ul, 262.6 ng/ul, and 189.3 ng/ul respectively) and sent to Plasmidsaurus.

Liquid culture of 36 and 37 (3 each from the plates streaked 8/22). Added arabinose to the 36 cultures (1%, so 500 uL). Plated transformations of 1, 5, and positive control at 10^0 and 10^-1 on amp plates.

8/24, CLO: Plasmid prepped 37A, 37B, 37C, and 36A. The concentrations were:

• 37A: 162.0 ng/ul
• 37B: 231.5 ng/ul
• 37C: 221.9 ng/ul
• 36A: Contaminated

8/29, JDS: PlasmidSaurus results arrived. Used Geneious software to visualize the sequences Plasmidsaurus sent back. P21 and p28 looked identical to the expected sequences barring 2-3 SNPs. p23 and p26 showed sequences that appeared to be undigested DH5a competent cells. This tells us that at least 2 of our GG constructs assembled correctly and should have functioned properly. The fact that we did not see any red on the meffRed constructs and we did see color on the mScarlet controls suggest that there is something wrong with the meffRed sequence.
On second review p21 and p28 are also not perfect. They have a section of IS1 that should have been excised during GG. In Conclusion - The problem is likely the GG Cloning, either it did not digest correctly or the cloning did not assemble correctly. The IS1 sequence could for some reason be interfering with the color for some reason too.

9/6, DTJ: Golden gate reactions were done for samples 37, 55, 60, and 62. After the reaction was completed on the thermocycler all the samples along with DVK were transformed into DH5Alpha competent E.Coli created in the lab. Samples were then shaken at 37°C for 2.5 hours before being plated at 10^0, 10^-1, and 10^-2. All 15 plates were incubated overnight at 37°C.

9/7, JDS:

Transformation results:

All of the plates had a small number (5-10) of big white colonies that finished in frequency with the dilution; these are likely KmR mutants of the competent E. coli. p37 worked great as a positive control. There were hundreds of small pink colonies on the 10^0 plate. he only other transformation with any colored colonies was p60 (the mScarlet test); it only had ~50 small pink colonies on the 10^0 plate however, so the efficiency of the cloning was lower. p55 also had the same colony types as the successful transformations, but no colored colonies. MeffRed may take more time to develop. p62 only had the large colony types. The cloning may have went wrong here and that is why no small potentially blue colonies are present. The 10^-1 dilution plate of each transformation was placed in the fridge, the other plates were all left in the 37°C incubator to determine if color would eventually develop.

Lab work:
Three each colonies from p55 and p60 were selected to be streak purified. Three pink colonies were selected from the p60 plate. Three small white colonies were selected from the p55 plate (hoping they turn red/pink eventually). Each strain was struck onto LBA+Km plates divided into thirds.

9/13, DTJ & JDS: Diluted CD parts (16), (17), (18), and (19) from dry pellet in tube to 20 ng/ul. Golden Gate cloned (16), (17), (18), and (19).
Transformation:
Followed the transformation protocol on the back of the NEB 5-alpha Competent E. coli (High Efficiency) with a few differences: Used our own competent cells (made 7/19/22), added 2ul of GG reaction to the competent cells, and shake at 37C for 3 hours. Plated 10^0 and 10^-1 dilutions of the 5 GG products and 1 DVK_AE positive control. Plates were left to incubate at 37°C overnight.

9/14, JDS & CLO: Poured 13x plates of LBA+Km^25. Melted the LBA made on 8/9/22 and added 175ul of Kanamycin stock (50mg/ml) to the 350ml of LBA.

All transformation plates appear successful. Each 10^-1 plate had ~100 colonies except for the DVK_AE control which was much more dense (~1000 on the 10^-1 plate). This is likely due to a higher amount of plasmids in the DVK_AE plasmid prep than the GG reactions. No color yet as these are arabinose inducible.
Three large colonies were selected from each of the five 10^-1 GG transformation plates (pGEC046-pGEC050). These colonies were wedge struck onto LBA+Km25 plates divided into thirds then left to incubate at 37°C overnight.

9/15, MJV:

Started liquid cultures of the new GoldenGate Products (pGEC046-050) from 9/14. Left to incubate at 37°C overnight.

9/16, CLO: Only pGEC046 and pGEC050 were pink. Plasmid prepped 46C and 50A.

9/20, MRH & DTJ & JDS: 5mL cultures of samples 46, 47, 48, 49, 50, and 60 were made as well as a negative control with no culture. 1.8L LB media was autoclaved and kanamycin was added. 50mL of 10% w/v arabinose was made, filtered, and placed in the freezer.

9/21, DTJ & CLO & JDS: Sample 60, his-tagged mScarlet with no CBD, was pink after overnight growth as it has a constitutive promoter. The negative control had no growth and the other samples were cloudy. 1mL of sample 60 along with 46, 47, 48, and 49 were then added to 250mL LB with kanamycin. 10ml of Arabinose was added to the cultures once they reached a DC600 between 0.6 and 0.8 (recorded below).

• 46: 0.766
• 47: 0.720
• 48: 0.829
• 49: 0.844

Three hours later none of the cultures were pink. 50ml of 10% w/v arabinose solution was made (5g of arabinose in water) and filter sterilized. Another 10ml of Arabinose was added to each 250ml culture and allowed to shake at 37°C overnight.

9/22, CLO: None of the 250ml cultures from yesterday were pink in the morning. Not even the constitutive. Dr. Geddes thinks there might have been contamination.

9/26, CLO & DTJ & JDS: Sent the Plasmid Preps of pGEC046 and pGEC050 from 9/16/22 to PlasmidSaurus. 1.8L LB media was made and autoclaved, 100mL 10% w/v arabinose solution was made and filter sterilized, and used 250mL flasks were autoclaved for the first time.

9/28, MRH: Acid washed the 250mL flasks.

9/29, DTJ & CLO & JDS: 100 mL cultures of samples 46, 48, 49, 50, and 60 were started from the overnight liquid cultures. After excess growth, the cells were diluted enough to have an optical density of 0.7 and arabinose was added. After 3 hours of incubation with arabinose, only the constitutively expressed sample (60) was pink. To address this, 50mL of each culture was removed and centrifuged to form pellets while the cultures were once again diluted to 150mL with additional arabinose and left to grow overnight.

9/30, DTJ: After growing overnight, inducible sample 50 was visibly more pink than constitutive sample 60 even though both express the same protein. Sample 48 was not pink at all, Sample 49 was slightly pink, and sample 46 was visibly very close in vibrance to sample 50. All cultures were centrifuged to form pellets and stored at 4°C.

10/3, JDS: Liquid cultures of pGEC047A, pGEC048C, and pGEC049C were started in LBB+Km25 and shook at 37C overnight. Fresh plates of pGEC046C, pGEC047A, pGEC048C, pGEC049C, and pGEC050A were streaked on LBA+Km25. Left in the 37°C incubator overnight.

10/4, DTJ: Plasmid prepped pGEC047A, pGEC048C, and pGEC049C.

10/5, CLO: Sent plasmid preps from yesterday off to plasmidsaurus.

10/7, DTJ & WW: Cell pellets 46, 47, and 50 were lysed using the ThermoFisher Scientific B-PER lysis kit. Despite most of the protein being insoluble, the supernatant was removed from each sample for testing. Sample 50 had the most soluble protein. In order to have direct comparisons between the different constructs, serial dilutions of sample 50 were performed and fluorescence was measured. A 1:16 dilution of sample 50 was found to be roughly equivalent to 46 and 47. Dyeing a t-shirt resulted in very weak visual results. Since 46 was the most promising sample, it and the 1:16 dilution of 50 were compared by creating 3 replicates of cotton t-shirt pieces that were dyed with 1mL of each solution. They were incubated at room temperature overnight in falcon tubes.

10/8, DTJ & WW: Samples from the previous night were imaged using a trans-illuminator. Then 40mL water was added to each sample and shaken at 250rpm for 30 minutes as a standardized wash procedure. Finally, the samples were re-imaged on the trans-illuminator.

10/9, JDS: PlasmidSaurus Results:

• pGEC046 = as expected - AraC+6xHis+mScarlet+Linker+CBDfimi+B0015+DVK
• pGEC047 = as expected - AraC+6xHis+mScarlet+Linker+CBDclos+B0015+DVK
• pGEC048 = Incorrect - - AraC+CBDfimi+Linker+meffRed+6xHis+B0015+DVK
• pGEC049 = as expected - AraC+CBDclosr+mScarlet+6xHis+B0015+DVK
• pGEC050 = as expected - AraC+mScarlet+6xHis+B0015+DVK

When designing the synthesized (16) CD gene fragment, the meffRed sequence must not have been changed to mScarlet as was intended. pGEC048’s incorrect sequence explains why it had almost no pink color in culture.

10/10, JDS: Liquid Cultures of the following strains were started in LBB+Km^25:
pGEC046, pGEC047, pGEC048, pGEC049, pGEC050, and pGEC060.

10/11, JDS: The following strains were stocked in the -80C freezer in an 8% DMSO solution:
pGEC046, pGEC047, pGEC048, pGEC049, pGEC050, and pGEC060.