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
• Submitted synthesis requests

Thursday, June 2

• meeting with life sciences people explaining project

• single sided synthesis with phosphotase direction?

• made amp plates:

• 100 mg / ml amp

• made liquid culture:

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

• spun overnight

Friday, June 3

• got our synthesis order in
• did a 2nd large materials order
• met with barrett funding person
• meeting with Jon, grad student from Misera'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 spun cells from yesterday
• spun for 2 hours in 37

Saturday, June 4

• made 500 ml SOC following protocol
• transformed resuspended dna into e coli

• followed transformation protocol, but did not use water bath

Monday, June 6

• transformed cells from saturday not growing
• test if competency procedure killed cells:

• make lb=

• repeat transformation with water bath instead of direct heat:

• thaw competent cells on ice
• 50 ul cells + 1 ul resuspended DNA, on ice for 30 minutes
• heat shock 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 (did not work):

• BBa_E0840
• BBa_E0240

• control onto non amp plate to test if transformation killed cells (it did not)

Wednesday, June 8

• autoclaved everything
• made 200 ml new SOB, 50 ml SOC
• new transformation:

• 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

• 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
• Transform that
• 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

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
• Made glycerol stock (ask Dan about procedure)
• Bought competent cells

• (top10)

• Got account set up (still need to create Sunrise account)

• Got Xiao refunded

• Met w/ James Alling, JD-PhD interested in helping us out

• He is very good at speaking and could help w/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 down total to 3.8kb instead of over 5kb
• We will try both ways, see if cas 1,2 do anything interesting

• primers for every CAS gene?
• Biobasic is taking twice as long as they advertised (no DNA until june 20?)

• From now on we will go through IDT
• Have contacted them about discount, will see what they say

Saturday, June 11

HAPPY 22ND BIRTHDAY KEITH!

Tuesday, June 14 - Friday, June 17

• Synthetic Biology 5.0 conference

Monday, June 20

• today:

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

• CCMB80

• tomorrow:

• genome prep (k12)
• pcr
• competent cells

Tuesday, June 21

• competency:

• CCMB80 w/ BL21 cells

• ruben, ethan
• made 250 ml SOB
• 9 210ul tubes, -80
• plates made:

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

• TSS procedure w/ BL21 cells

• madeline, juan, keith
• 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:

• plates made:

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

• genome prep:

• nisarg

• First PCR overnight of CAS genes

Wednesday, June 22

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

• don't know if amp was any good

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 of 2, 6, 7, 8, 11 (3 each in LB + amp broth)
• 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
• overnight culture in amp grew
• today:

• glycerol stock of DA
• overnight cultures of DB, sEQ1 from plates
• 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

• gel from pcr last night

• still doesn't work!

• dna extraction using spin method (DB, SEQ1)

• still doesn't work!

• 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 in the bb ampr vector into tsp and neb was successful
• made overnight culture to miniprep tomorrow

Sunday, June 26

• lb amp plates
• restriction digest

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

• ran gel on previous

• didn't linearize plasmid before gel

Monday, June 27

• top 10 lab techniques to learn and love

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

• redid restriction digest

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

• gel errors

1) did not let gel dry completely before removing comb

2) too much voltage caused gel deformation

• ran out of PSB1A3

• lesson: don't use it directly! must grow it up first
• ordered more from igem hq

• cultured b halodurans

• rehydrated and let culture overnight in tryptic soy broth

• overnight culture 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

• from overnight culture

• made 4 plates (tryptic soy), as well as 7 more tryptic soy plates
• glycerol stock x 1

• 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

• Miniprep of Seq1, DA, DB, E0840
• Nanodrop results (see Kylie's notebook)
• Restriction digests

• Made "master mix" of water, BSA, NEB4 (1x, enough for 30 digests)
• Seq 1: ES, EX
• DA: ES, EX
• DB: ES, EX
• E0840: EP
• Gel (large size)
• 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 inventory (mostly)
• 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!
• Tomorrow: plan on ligation, check PCR, run gel for PCR, order primers for 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 (2 x)
• run gel: CMR product from BH PCR

• gel extraction, submitted for sequencing

• very low yield (~20 n)

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 got what we want
• Gel results:

• Cultures of RA/RB grew well
• Miniprep of RA x2, RB x2

• Restriction:

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