Team:Stanford/Notebook/Lab Work/Week 2

7/5 Monday

University Holiday: Planning Meeting moved to tomorrow

7/6 Tuesday

Planning Meeting Notes

Agenda

• Design Updates
  • Analog Comparator (Chris & Alex)
  • Redundancy between the two proposals
  • Degree of phosporylation
  • Dynamic range and mechanisms

• • Digital Comparator (Francisco & Karina)
  • RSID; standardizing the design
  • Introducing a PoPs signal output

• Characteristics between the two Comparators
  • What makes the two different?

• Applications
  • Vaginal Infections/Preterm Birth

• is it really a ratio?
• Other ideas: Targeting Cancer Cells, oscillators

• Administrative Details
  • Ordering Stuff: Oligos ordering sheet, Digestion and Gel Extraction Kits?
  • Stipend?
  • Sponsorship Updates
  • MDV Presentation

Goals For the Week

• Chris & Alex
  • need to make 50x TAE
  • complete plasmids as far can get w/o kinase and phosphotase
  • order DNA sometime this week
• Francisco & Karina
  • Run orginal and new designs by Christina
  • design standard sRNA designs and place order by the end of the week

2:00 Meeting with Christina (Karina, Francisco, Laura)

to test: sRNA complementary to ID-RBS-scar; another sRNA centered across RBS as control (to see if shifting will affect efficiency of sRNA)

To Do:

1. check registry for shorter pBAD promoters? PCR own modified version?
2. finalize ID's

• RNA folding: avoid local strong structures, > -10kcal/mol good: look at free energies of individual hairpins
• BLAST to make sure not in E. Coli genome

Alex's Notebook

• Restriction Digests:
  • Take the part out completely, and/or take out either prefix or suffix.
  • KEEP SOME DNA FOR GEL CONTROL!!!

• 2K3 E/P
• 1A2 E/P
• E0240 1A2; X/P length: 876
• T9002 1A3; E/P length: 1945

Greg's Notebook

• During weekly meeting got up to speed with what the team’s been doing while I was away
• Used heat-shock transformation protocol for DH5alpha to transform with the following genetic material, left to culture overnight:

Function	Part #	Distribution Location	Resistance
ribosome binding site	B0032	P1 2I	Amp
forward double terminator	B0015	P1 23L	Amp + Kan
bidirectional double terminator	B0014	P2 24C	Amp + Kan
medium constitutive promoter	J23107	P1 20A	Amp
strong constitutive promoter	J23100	P1 18C	Amp
plasmid backbone	pSB1K3	P1 5A	Kan

Chris's Notebook

7/6/2010 Goals for Today: Restriction Digestion of Promoters (pBAD and F2620), GFP reporter (E0240), and plasmid. Ligation of promoters, reporters, and plasmids. Chris:

      4A5     E/P 3395 bp
      I0500   E/S (on psb2K3) 1210 bp
      F2620   E/S (on pSB1A2) 1061 bp

Ryan’s Protocol for Restriction Digest Cloning Digestion (50μl reaction volume)

• Plasmid 12.0 μl
• Each enzyme 1.0 μl
• NEB buffer 5.0 μl
• 10x BSA 0 or 5 μl (optional depending on enzymes used)
• NP water Bring to 50 μl total volume (26 ul)
• Let sit for at least an hour at 37 degrees C

Heat Kill for 20 minutes at 65 degrees C

• Note: Jerome’s I0500 is extremely concentrated (1.25 ug/ul).
• Restriction Digest ran overnight until 940 am the next morning

7/7 Wednesday

Greg's Notebook

• Checked plates from previous day's transformation
  • B0032 and B0014 showed little growth
  • pSB1k3 showed fantastic growth, with distinct colonies covering the entire plate
  • B0015, J23100, and J23107 all showed normal growth

• poured plates, stored in cold room:
  • 22 Amp
  • 20 Kan
  • 18 Chlor

Francisco's Notebook

• Transformed E.coli with E1010 (RFP) using heatshock. Used 1uL DNA from distribution.
• Transferred 50uL of transformed cells to agar plate with kanamycin.

Chris's Notebook

Running a Gel of F2620, pBAD, and psb4A5:

• Make a 0.8% agarose gel (0.4 g of agarose in 50 ml of TAE)

-Heat in microwave until agarose is completely dissolved -Add 10.0 ul of EtBr to mix (5000x for a 50 ml solution)

• let run for approximately 1 hr
• refer below for bands:

    4A5     E/P 3395 bp
      I0500 (pBAD)  E/S (on psb2K3) 1210 bp
      F2620   E/S (on pSB1A2) 1061 bp

Making Kanamycin, Ampicillin Chloramphenicol Plates

Kanamycin is at 1000x

PCR of I746908 to isolate the RBS, CDS, and double terminators

Programming the Block:

• 1. 95 C for 2 minutes
• 2. 95 C for 30 sec
• 3. 55 C for 30 sec
• 4. 68 C for 1.10 min
• 5. go to step 2 for 30 cycles
• 6. 72 for 5 minutes
• 7. end (0=forever)

Use PCR HF SuperMix For each reaction:

  1.25 ul of forward primer
  1.25 ul of reverse primer
  1.0 ul of DNA template
  46.5 ul of PCR HF SuperMix

7/8 Thursday

Alex's Notebook

Prepared a bunch of restriction digests. Realized some parts are way too small to follow normal BioBrick assembly.

32, 34 (X/P), J0, J7, F2620, I0500 (E/S), 1A3, 4A5, 2K3, I8, and T9 (E/P).

Christopher's Notebook

Running a Gel of F2620, pBAD, and psb4A5:

• Make a 0.8% agarose gel (0.4 g of agarose in 50 ml of TAE)
• Heat in microwave until agarose is completely dissolved
• Add 10.0 ul of EtBr to mix (5000x for a 50 ml solution)
• let run for approximately 1 hr
• refer below for bands

      4A5     E/P 3395 bp
      I0500   E/S (on psb2K3) 1210 bp
      F2620   E/S (on pSB1A2) 1061 bp

Gel Extract the Fragments Running a Gel of PCR Product:

• first need to PCR cleanup
• then run on 1.4% agarose gel (0.7 g of agarose in 50 ml of TAE)
• this is necessary because of the 800 bp size of the fragment

Miniprep Ligations

• F2620+E0240+psb1A2
• pBAD+E0240+psb1A2

Restriction Digests PCR product F2620

Laura's Notebook

• miniprepped Alex's/Chris' liquid cultures, using Promega kit
  • 2 each of:
    • psb1K3
    • J23107
    • B0032
  • eluted in 100 uL H2O

Nanodrop Data
tube ID	260/280	260/230	ng/uL
psB1K3-1	1.77	1.08	71.3
psB1K3-2	1.73	1.01	77.8
J23107-1	1.87	1.60	116.3
J23107-2	1.90	1.74	142.0
B0032-1	1.81	1.20	68.1
B0032-2	1.83	1.32	72.5

Greg's Notebook

• Ran nanodrop of miniprepped stuff from yesterday:

Part #	260/280	260/230	ng/uL
B0034	1.68	1.65	26.7
I746908	1.86	2.91	59.4
J23100	1.01	2.03	91.4
pSB3c5	1.81	2.01	104.8

Francisco's Notebook

• Toothpicked a colony from the E1010 plate from yesterday. Inoculated in 4.5mL of LB + kanamycin.

7/9 Friday

Friday Recap Meeting

• [http://docs.google.com/present/edit?id=0Ae31et9w_tAhZGZyc3R2ejJfOGNnY2drMmZz&hl=en&authkey=CPHGzfIB Weekly Leader Presentation: Karina]

• Analog Comparator update: Chris

• MDV Presentation Updates & Planning
  • July 16, 2010 11:00 am
  • Separate Meeting for planning, Tuesday 4:00 pm, Green Atrium
  • Grad Students: Ryan

• Administrative Details

• Stipend?
  • Check on Axess for Billing statements
• Sponsorship Updates

• Christina’s announcements:
  • Start talking about Medal Requirements
  • Keep in mind the wiki!
  • Digitized Cloning Scheme

• Medal Requirements- Presentation next week how we’re addressing those
  • Collaborative, Improving Standard, Human Practice
  • Idea: improving promoter database on Parts Registry
  • Need to get access to parts registry to edit  e-mail Drew to e-mail them
  • Gold Medal Meeting Meeting Wed Morning at 9:00 am? Send out the Doodle (Anusuya)

• Next Weeks Leader: Alex

Francisco's Notebook

• Miniprepped E1010 plasmids from yesterday's liquid culture.
  • Nanodrop drop results: 260/280: 1.89; 260/230: 1.85; ng/uL: 282.4

Karina's Notebook

Today's Goal: Digest GFP, RFP, and Terminators. Will do (GFP + T) and (RFP + T) ligations on monday.

Part #'s:

• GFP: E0040 (cut at S and P)
  
• RFP: E1010 (cut at S and P)
  
• Term: B1006 (cut at X and P)
  
  

Recipe

• 12.0 uL DNA
  
• 1.0 uL each enzyme
  
• 5.0 uL NEB buffer 2
  
• 5 uL BSA (10x)
  
• Sterile H20 to fill up to 50 uL
  
  

Mix and put 37º waterbath for 2 or more hours.

Making Gel
To make 50 mL of 0.8% agarose gel, add:

• .4 g agarose
  
• 50 mL TAE (1x)
  
• 10 uL EtBr
  
  

Loading the Gel

• add 10 uL loading dye (6x) to digests
  
• add 40 uL of dye + digests to wells
  
• don't forget to include wells with controls! (uncut plasmids)
  
• load 10 uL ladder
  
• run at 95 V for about an hour
  

!!note sizes of plasmids/inserts
GFP plasmid (pSB1A2) --> 2079 bp
GFP insert --> 720 bp
RFP plasmid (pSB2K3) --> 4425 bp
RFP insert --> 681 bp
Terminator plasmid (pSB1AK3) --> 3189 bp Terminator insert --> 39 bp

Gel Results

All bands look fine except for, there were no bands for the terminators. They were so small they ran off the gel. We are considering using PCR purification to isolate these parts.

Gel Extraction
[http://molecool.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf Follow QIAquick Gel Extraction Kit Protocol], skip isopropanol step and elute in 50 uL H20.

Gel Extraction
Part	Tubes (g)	Gel Extract + tube (g)	Gel Extract (g)
GFP	1.06	1.11	.05
RFP	1.09	1.22	.13

Laura's Lab Notebook

• worked with Karina and Francisco on digestions, gel, gel extraction (see Karina's notebook for details)
• for digestion: Francisco set up RFP, Karina set up terminator, I set up GFP

Chris's Notebook

Completion of PCR

Note: only use PCR cleanup if the DNA is going to be used for ligation

Running a Gel of PCR Product:

• first need to PCR cleanup
• then run on 1.4% agarose gel (0.7 g of agarose in 50 ml of TAE)
• this is necessary because of the 800 bp size of the fragment
• run at 85 volts
• small combs

Restriction Digest of I746908-E/P

• Cloning Digestion (50μl reaction volume):

      4A5     E/P 3395 bp
      I0500   E/S (on psb2K3) 1210 bp
      F2620   E/S (on pSB1A2) 1061 bp
      3C5     E/P 2738 bp (buffer 3)
      F2620   E/S (on pSB1A2) 1061 bp

Run Gels of Restriction Digests

• run on 1.4% agarose gel (0.7 g of agarose in 50 ml of TAE)
• this is necessary because of the 800 bp size of the fragment
• run at 85 volts
• small combs

Expected Sizes:

   3C5     E/P 2738 bp (buffer 3)
   F2620   E/S (on pSB1A2) 1061 bp
   I746908 E/P (on psb1A2) 2093 bp
   B0034 X/P (on psb1A2)  12 bp-do NOT run on gel
   J23100 E/S (on psb1A2) 35 bp-do NOT run on gel

Gel Extract

7/10 Saturday

Run gels for the restriction digests. 2 failed due to similar sizes Could not ligate current pieces.

New Reporter

• I746916 (N/A)

7/11 Sunday

Prepared new triple restriction digests with EcoRI, PstI, and BglI.

• I8 and T9.

Inoculated 3C5, 1K3, I0500, and I8 to obtain more DNA.