IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/06

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Team Allergy

Today, we isolated plasmid that contained antisense and sense sequences of the allergen panel and introns PME and PAL that we grew last week.

Procedures

For amiRNA

  1. Nanodrop of miniprepped amiRNA plasmids
  2. Diagnostic digest of amiRNA plasmids
  3. E Gel of digested amiRNA
  4. Send plasmids to Genewiz for sequencing

For ihpRNA

  1. Miniprep of plasmids containing introns for hpRNA
  2. Nanodrop of miniprepped intron plasmids
  3. Miniprep of plasmids containing Allergen Panel sense and antisense
  4. Nanodrop of minipreped allergen plasmids
  5. Diagnostic Digest of introns and allergens
  6. E Gel of digested parts
  7. Extract pKannibal and pHannibal vectors and transform into E. coli
  8. PCR of the PDK intron in pKannibal and pHannibal

Results

For amiRNA

Miniprep of turbo bacteria for plasmids containing sequences for amiRNA

Should obtain plasmids containing the complete sequence for amiRNA. The resulting concentration should be relatively high (<100ng/μL) because all growing bacteria would have the plasmids inside.

Nanodrop of miniprepped amiRNA plasmids

We used 2μL of DNA per nanodrop.

Concentrations in ng/μL
Sample # --> One Two Three
GFP 134.4 167.6 207.2
GFP 0.5 163.3 106.9 230.9
LTP 134.0 127.8 165.5
LTP 0.5 158.5 137.2 113.5
Bet 129.4 78.9 124.4

Diagnostic digest of amiRNA plasmids Digested 200~500ng of plasmids with EcoR1 and Spe1. We will then run the digested vectors on an E Gel and look for DNA that is the length of the insert. If such DNA appears on the gel, then we will send it off to be sequenced.

E Gel of digested amiRNA

Image of E Gel plates two and one

We had to run 21 samples, so we used two gels. The orders were:

Gel #1

  1. 1kb Ladder
  2. GFP 1
  3. GFP 2
  4. GFP3
  5. GFP 0.5 1
  6. GFP 0.5 2
  7. GFP 0.5 3
  8. LTP 1
  9. LTP 2
  10. LTP 3
  11. LTP 0.5 1 no visible band for neither plasmid nor insert
  12. LTP 0.5 2

Gel #2

  1. 1kb Ladder
  2. LTP 0.5 3
  3. Bet 1
  4. Bet 2
  5. Bet 3
  6. Neg Control for hpRNA (digested with Xba and Pst, and ligated without new inserts) no visible insert band
  7. Neg Control for intron (digested with Eco and Spe, and ligated without new inserts)
  8. PME Good no visible insert band
  9. PME Dubious no visible insert band
  10. PAL Good colony 1 no visible insert band
  11. PAL Good colony 2
  12. 1kb Ladder

For ihpRNA

Miniprep of plasmids containing introns for hpRNA Nanodrop of miniprepped intron plasmids

We used 2μL of DNA per nanodrop.

Concentrations in ng/μL, cells are filled when applicable
Insert Name Good Dubious Colony 1 Colony 2 Colony 3
PME 85.6 66.8
PAL 67.7 108.8
Neg Control, Intron 74.0
Neg Contron, hpRNA 63.5 30.1 37.3
  1. Miniprep of plasmids containing Allergen Panel sense and antisense (from overnight cultures)--still need to do preps of cultures grown today
  2. Nanodrop of minipreped allergen plasmids
  3. Diagnostic Digest of introns and allergens
  4. E Gel

Gel #1

Lane 1: 1kb Ladder

Lanes 2-4: Ger Sense

Lanes 5-7: Ger Antisense

Lanes 8-10: LTP Sense

Lanes 11-13: LTP Antisense

Lanes 14-16: Bet 2 Sense

Lanes 17-19: Bet 2 Antisense

Lanes 20-22: Bet 1 Sense

Lanes 23-25: Bet 1 Antisense


phannibal and pkannibal Extracted vectors from a piece of paper per instructions. Transformed vectors into E. coli. Miniprep and PCR to extract PDK intron.

  • pcr to get out pdk intron

Team Fence

Inoculated 7 Barstar and 3 NLS colonies from Thursday's ligation.

Performed PCR on LacIn (E10) following standard PCR measurements as previously described.

Digestion of B21 backbone using Xba1 and Pst1

Total volume per reaction: 50 μL

  • 2μL Xba1
  • 2μL Pst1
  • 10μL B21 plasmid (0.3μg/μL X 10μL = 3μg DNA)
  • 5μL 10X digestion buffer
  • 31μL H2O

File:76B21 digest gel.pdf

Digestion successful, larger bands removed for gel extraction.

Gel extraction performed according to QIAgen gel extraction protocol.

Team Flavour

Tagging Miraculin and Brazzein with YFP and StrepII tags

Tag: -E--N--X--STREP/YFP--S--N--P-
Insert: E--N--X--Miraculin/Brazzein--S--N--P-

  • The 'tag' biobrick (either YFP or STREPII) was used as the vector; Miraculin and Brazzein were used as the insert.
  • Tags were ligated to both the N- and C-terminal of Miraculin and Brazzein

BBa_J45700 BBa_J45700

BioBrick Digest Combinations
Vector Insert
N-Terminus Spe1/Pst1 Xba1/Pst1
C-Terminus EcoR1/Xba1 EcoR1/Spe1


Digestion Reactions
B21 S/P B21 E/X Miraculin X/P Miraculin E/S Brazzein X/P Brazzein E/S
diH2O 13 13 11 11 0 0
Green FD buffer (10x) 2 2 2 2 2 2
DNA 3 3 5 5 16 16
Spe1 1 - - 1 - 1
PstI 1 - 1 - 1 -
EcoR1 - 1 - 1 - 1
Xba1 - 1 1 - 1 -

Gel

The gel showed DNA fragments consistent with Miraculin and Brazzein. The digestion of B21 appeared to be succesful, but the DNA sequence cut out was too small to see on the gel.

Miraculin brazzein b21.jpg

   Gel Lanes:
    1. 1 kb plus ladder
    2. B21 speI/pstI
    4. B21 ecoRI/xbaI
    6. Miraculin xbaI/pstI
    8. Miraculin ecoRI/speI
    10. Brazzein xbaI/pstI
    12. Brazzein ecoRI/pstI
    14. 1kb plus ladder


We extracted the circled bands and gel purified the gel per the Qiagen gel purification protocol.

   ODs of gel purified DNA
    B21 speI/pstI:  15.9 ng/μL
    B21 ecoRI/xbaI:  11.0 ng/μL
    Miraculin xbaI/pstI:  9.7 ng/μL
    Miraculin ecoRI/speI:  7.1 ng/μL
    Brazzein xbaI/pstI:  2.6 ng/μL
    Brazzein ecoRI/pstI:  5.0 ng/μL


Ligation

We did 6 different ligation reactions. The chart below shows the different reactions. Ligation reactions were left at room temperature for 15 minutes.


Ligation Reactions
Miraculin xbaI/pstI w/ B21 speI/pstI Miraculin ecoRI/speI w/ B21 ecoRI/xbaI Brazzein xbaI/pstI w/ B21 speI/pstI Brazzein ecoRI/speI w/ B21 ecoRI/xbaI Control B21 speI/pstI Control B21 ecoRI/xbaI
DNA Insert 2 3 3 1 0 0
T4 DNA ligase buffer (10x) 2 2 2 2 2 2
diH2O 12 9 11 11 14 12
T4 DNA ligase 1 1 1 1 1 1
DNA Backbone 1 1 1 1 1 1


Transformation

We mixed 5μL ligation mix with 15μL Turbo e. coli cells. These were placed in ice for 30 minutes and then heat shocked in 42°C water bath for 30 seconds. The cells were placed back on ice for 2 minutes. 170 μL of SOC broth was added to each Eppendorf tube. The transformed e. coli were then plated on LB Ampicillin plates and left in the 37°C incubator overnight.