Team:Arizona State/Notebook/June

Wednesday, June 1

• Made agar with 3.7 g / 100 ml DI water
• Made 2 plates from 2 MG1655 strains received yesterday from Misra
• Submitted synthesis requests for RA, DA, and Seq1 from BioBasic

Thursday, June 2

• Meeting with Dr. Chang and her grad students from the School of Life Sciences to explain iGEM project.

• Single sided synthesis with phosphotase direction?

• Made amp plates:

• 100 mg / ml amp

• Made liquid culture using LB Broth:

• 5 g peptone
• 2.5 g yeast extract
• 5 g NaCl
• 500 ml water

• Incubated and shook liquid culture overnight

Friday, June 3

• Confirmed placement of synthesis order
• Placed second large order for necessary laboratory materials
• Met with Barrett funding advisor
• Met with Jon, grad student from Misra's lab
• Lab:

• Resuspended part E0840 from well following parts registry protocol
• Followed "competent cells and chemical transformation procedure for DH5 alpha":

• Made 20mM concentration MgCl2 in shaken cells from yesterday
• Shook for 2 hours in 37 C room

Saturday, June 4

• Made 500 mL SOC following OpenWetWare protocol
• Transformed resuspended DNA into E. Coli

• Followed transformation protocol, but did not use water bath

Monday, June 6

• Transformed cells from Saturday, June 4th did not grow yet
• Test if competency procedure killed cells using following procedure:

• Make LB=

• Repeat transformation using water bath instead of heat block:

• Thaw competent cells on ice
• 50 ul cells + 1 ul resuspended DNA, on ice for 30 minutes
• Heat shock cells at 42 in a water bath for 60 seconds
• Incubate on ice, 5 min
• Add 100 ul SOC to cells
• Shake at 37 C for 2 hours (11 am - 1 pm)
• Plate 20 ul, 200 ul (2 plates)
• Incubate overnight

Tuesday, June 7

• Amp plates did not grow
• Competent cells plated without amp grew
• New transformation using 2 different parts conducted did not work with either part:

• BBa_E0840
• BBa_E0240

• Control onto non amp plate to test if transformation killed cells showed that the cells were still viable

Wednesday, June 8

• Autoclaved lab materials to be sterilized
• Made 200 ml new SOB, 50 ml SOC
• New transformation protocols:

• Top10 chemically competent E. Coli from biodesign
• Part: BBa_E0840
• Using top10 protocol
• Plates: # 4 50ul, 5 150ul

• MG1655 plated from plate # 2:

• Plate: # 1
• From: 6-2 plate MG1655

• Overnight culture:

• From plate # 3

Thursday, June 9

• Xiao introduced 3 grad students who can offer advice/assistance throughout the project

• We will meet with them (likely Thursdays @ 10am) to update them on our progress

• Yesterday's plates:

• #4, 5 have colonies but no glow with UV - no promoter in biobrick part

• Need to add in a promoter

• Today:
• Add in promoter for GFP construct

• Constitutive promoter:

• Part: BBa_J23101

• Use Knight restriction protocol

• Cut promoter BBa_J23101 with ECORI, SPEI
• Cut GFP generator BBa_e0840 with ECORI, XHOI
• DNA extraction- use "ethanol precipitation of nucleic acid" procedure

• Ligate restriction products
• Transform ligation products
• Create stock of competent cells

• Order Top10 cells (what strain are these?)
• Make glycerol stock of BioBrick
• Jon/Misra procedure for competent cells and transformation:

• Overnight culture from previous:

• Diluted 1 to 50
• Shook 1 hr in 37 C room

Friday, June 10

• Lab today:

• 2 competency procedures (Jon, CCMB80)
• 3 transformation procedures (Jon, CCMB80, top10 from biodesign)
• 12 plates made (see lab notebook)

• Autoclaved glass test tubes
• Dan demonstrated to lab members how to make glycerol stocks
• Bought top10 competent cells
• Got account set up (still need to create Sunrise account)

• Got Xiao refunded

• Met with James Alling, who is a JD-PhD interested in helping us out

• He is very good at speaking and could help with presentation later
• Very attracted to promoting big picture of project

• Kylie determined new primers after noticing that we don't need Cas 1,2,3 for our natural cas construct (from "structural basis for CRISPR")

• This brings Cas construct size down to a total of 3.8kb instead of over 5kb
• We will try both ways, and see if cas 1 and 2 do anything interesting

• We are considering getting primers for each individual Cas gene (Cas A, Cas B, etc...)
• Biobasic is taking twice as long as they advertised (no DNA until june 20?)

• From now on we will go through IDT due to slow turnaround from BioBasic
• We contacted BioBasic about discount, got a synthesis price reduction

Saturday, June 11

HAPPY 22ND BIRTHDAY KEITH!

Tuesday, June 14 - Friday, June 17

• Synthetic Biology 5.0 conference

Monday, June 20

• today:

• Prepared overnight culture x 2 (LB) for DNA miniprepping
• Prepared overnight culture x 2 (LB, SOC) for culture

• CCMB80

• Tomorrow:

• Carry out genome prep for K12 genome from MG1655
• Begin PCR amplification of Cas genes
• Create and test competent cells

Tuesday, June 21

• Competency information:

• CCMB80 w/ BL21 cells

• Ruben and Ethan carried out this competency procedure
• Made 250 ml SOB
• 9 210ul tubes placed into -80 C fridge
• plates made:

• 1. LB, transformation: DA
• 2. LB + amp, transformation: DA
• 3. LB + amp, transformation: PUC19

• TSS procedure w/ BL21 cells

• Madeline, Juan, and Keith carried out this competency procedure
• plates made:

• 4. LB + amp, PUC19, burned
• 5. LB + amp, PUC19, unburned
• 6. LB + amp, DA
• 7. LB + amp, DA
• 8. LB + +amp, DA

• NEB top10 competency test:

• Plates made:

• 9. LB + amp, PU19
• 10. LB + amp, PUC19
• 11. LB + amp, DA

• Genome prep:

• Carried out by Nisarg

• First PCR conducted overnight of attempting to amplify CAS genes A-E and 3

Wednesday, June 22

• Made new LB + amp stock
• Made 200 ml LB + amp broth
• Results from plates made yesterday:

• Ampicillin stock integrity in question

1: normal growth (no distinct colonies)

2: colonies

3: no growth

4: no growth

5: no growth

6: colonies

7: colonies

8: colonies

9: very heavy colonies

10: very heavy colonies

11: light colonies

• Overnight cultures made of 2, 6, 7, 8, 11 (3 each in LB + amp broth)
• Ran a gel of PCR product
• New plates:

1. CCMB80, LB, PUC19

2. CCMB80, LB, DB

3. CCMB80, LB, SEQ1

4. CCMB80, LB + amp, no plasmid

5. CCMB80, LB + amp, PUC19

6. CCMB80, LB + amp, DB

7. CCMB80, LB + amp, SEQ1

8. NEB, LB, PUC19

9. NEB, LB, DB

10. NEB, LB, SEQ1

11. NEB, LB, no plasmid

12. NEB, LB + amp, no plasmid

13. NEB, LB + amp, PUC19

14. NEB, LB + amp, DB

15. NEB, LB + amp, SEQ1

16. TSS, LB, PUC19

17. TSS, LB, DB

18. TSS, LB, SEQ1

19. TSS, LB, no plasmid

20. TSS, LB + amp, no plasmid

21. TSS, LB + amp, PUC19

22. TSS, LB + amp, SEQ1

23. TSS, LB + amp, DB

Thursday, June 23

• Plates from yesterday worked completely as expected

Plate 6:

Plate 15:

• Overnight culture in amp grew
• Today:

• Glycerol stock made of DA
• Overnight cultures made of DB, sEQ1 from plates
• Ran gel of PCR from last night
• DNA extraction 2x (elution)- verified using nanodrop, did not get enough to be successful
• Another overnight PCR using different settings
• Designed new primers for casA-E + cas3

Friday, June 24

• Ran gel from PCR carried out last night

• Still doesn't work!

• DNA extraction using spin method from Miniprep protocol(DB, SEQ1)

• Still doesn't work!

• We went through hassle of ordering new Cas primers from IDT

Ordered a pair of primers for each Cas gene - this way we can customize and perhaps PCR out in sections

Saturday, June 25

• Transformations of DA, DB, and Seq1 into the BioBrick ampR vector (pSB1A3) into TSS and NEB cells was successful
• Made overnight liquid culture to miniprep tomorrow

Sunday, June 26

• Made LB amp plates
• Conducted restriction digest...

• We used the wrong enzymes! used EX and EP instead of EX and ES

• Ran gel on previous PCR

• Didn't linearize plasmid before running results on a gel

Monday, June 27

• We identified the Top 10 lab techniques to learn and love

• Dan emphasized that we need to be independent and know these!

• Redid restriction digest

• Two methods for restriction: Ginkgo bioworks (two bricks into desired plasmid) and traditional (EX and ES)
• DA: ES, EX
• Seq1: ES, XP
• PSB1A3: EX, EP

• Some Gel errors

1) did not let gel dry completely before removing comb

2) too much voltage caused gel deformation

• Ran out of PSB1A3

• Lesson learned: don't use it directly! must grow it up first
• Ordered more from iGEM HQ

• Cultured B. Halodurans

• We rehydrated cells and let culture grow overnight in tryptic soy broth

• Made overnight cultures of Seq1, DA, DB, and E0840
• Overall message: Not a great day in terms of results, but many tough lessons learned.

Tuesday, June 28

• B. halodurans developments

• Cells retrieved from overnight culture

• Made 4 plates on tryptic soy media, as well as 7 more tryptic soy plates
• Made one glycerol stock

• Conducted Genomic prep + PCR using primers R1 and R2

• Nanodrop new record! 220ng/ul template DNA
• "Ode to Trinette" Haiku by Joseph Flay

PCR is hard

Trinette, you are so thermal

Thanks for the fun times

• Conducted miniprep of Seq1, DA, DB, E0840

• Nanodrop results (see Kylie's notebook)

• Further restriction digests

• Made "restriction supermix" of water, BSA, NEB4 (1x, enough for 30 digests)
• Seq 1: ES, EX
• DA: ES, EX
• DB: ES, EX
• E0840: EP
• Made and ran a large gel
• Problem: used wrong hyperladder (used I instead of II)

• Successfully isolated: Seq 1 (ES), Seq 1 (EX), DB (EX), E0840 (insert), E0840 (vector)
• Unsuccessful: DA (ES), DA (EX), DB (ES)
• Transformed RA, RB into NEB cells (no control)
• Replated BL21DE3 x 1 and MG1655 x 1 on LB Agar
• Moved plates from 4 degree room to small fridge in lab because they are fixing the room tomorrow (and got rid of some old plates)
• Took lab inventory (mostly)
• Made more overnight cultures:

• B. halodurans x 1

• Other notes: Paul Johnson sent us a nice message basically saying that as long as we can justify it, iGEM is here to stay

  (meaning they will keep funding the team in the coming years)).

  We also talked about getting FURI and SOLUR funding for next year's team.

  An REU proposal was discussed, but ultimately abandoned because a majority of the team's students would need to be from outside ASU, which we don't want.

• Overall: people kept very busy, we worked well in teams, however we need to make sure we are really paying attention to what we do - mistakes cost time and money!
• Tomorrow: plan on ligation, check PCR results, run a gel for PCR results, order primers for B. halodurans, try restriction of DA again, miniprep and try restriction of RA/RB

Wednesday, June 29

• Plates from last night (see pictures):

• LB + AMP + RA
• LB + AMP + RA
• LB + AMP + RB
• LB + AMP + RB

• Restriction digest of DA, DB (2x)
• Run a gel: CMR product from BH PCR

• Conducted gel extraction, submitted extracted DNA for sequencing

• Very low yield (~20 ng/uL)

Thursday, June 30

• New primers arrived from IDT for second round of attempts at getting the cas genes out of MG1655
• After successful isolation of what looks like the CMR genes from Bacillus halodurans, a second attempt was run overnight
• Got our sequence data from last night for CMR genes: looks like we successfully amplified CMR!

• Gel results:

• Cultures of RA/RB grew well
• Conducted miniprep of RA x2, RB x2

• More restrictions:

• DA ES EX (2x)
• DB ES XP (2x)
• RA ES EX XP (2x)
• RB ES EX XP (2x)