IGEM:Harvard/2010/Team Vector

Open Series

Expression Series

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Reporter Series

Abstract

Our aim was to create a Biobrick standard vector to be used in plant engineering. To do so, we modified the pORE vector system created by Coutu et al, a system designed to facilitate agrobacterium-mediated transformation of plants. Working with two of each of the three basic types of pORE vector, we removed the existed multiple cloning site and replaced it with the Biobrick standard multiple cloning site.

6-14-2010

Started preparation of agrobacterium vector backbones

• Digested O1, O2, E3, E4, R1, R3 with HindIII and SpeI (Fast Digest Protocol - make link)
• Ran digests on 1% agarose gel (protocol link)
• Digest successful for O1, O2, and R1 but not for E3, E4, and R3 (reason unknown)
• 
• Cut out backbone (top) bands from O1, O2, R1 and saved them at 4°C

6-15-2010

Continued preparation of agrobacterium vector backbones

• Re-digested E3, E4, R1, R3 with HindIII and SpeI (Fast Digest Protocol - make link)
  • Ran digests on 1% agarose gel (protocol link)
  • Digest successful for all reactions
  • 
  • Cut out backbone (top) bands for E3, E4, R3 from the gel
• Realized that ordered O1, O2 oligos had SacII sticky and rather than HindIII
  • Re-digested O1, O2 with SacII and SpeI to prepare backbone with the correct sticky ends (regular digest protocol, buffer 4)
  • Ran digests on 1% agarose gel (protocol link)
  • Digest successful for both reactions
  • 
  • Cut out backbone bands for both reactions
• Annealed open series insert oligos (synthesized by Mr. Gene) (protocol link - karmella's)
• Gel purified bands for all reactions (protocol link)
• Realized that backbones needed to be dephosphorylated before ligation, so redid digestions with phosphatase.
  • Digested O1, O2 with SacII and SpeI, with TSAP (phosphatase) added (slow digest protocol, buffer 4, 30 ul reactions). Did two digest reactions for each vector to increase yields.
  • Digested E3, E4, R1, R3 with HindIII and SpeI, with TSAP added (fast digest protocol, 30 ul reactions). Did two digest reactions for each vector to increase yields.
  • Ran digests on 1% agarose gel (protocol link)
  • Digest successful for all reactions
  • 
  • Cut out backbone (top) bands for each reaction, keeping the two digests of each vector in the same eppendorf tube.

6-16-2010

Completed preparation of agrobacterium vector backbones and prepared expression series and reporter series inserts

• Gel purified digest slices from previous day (protocol link)
• Ran a PCR reaction on E3, E4, R1, R3 vectors with primers synthesized by Mr. Gene (PCR phusion protocol) to generate inserts
  • Melting Temperature: 60ºC
  • Elongation Time: E3, E4, R3 (~0.6 kb) - 15 seconds; R1 (~2 kb) - 30 seconds
  • Ran part of each reaction on 1% agarose gel to determine if correct products were formed
  • PCR reactions were successful
  • 
  • PCR purified remainder of each reaction
• Digested PCR products to prepare products with correct sticky ends (two digests per vector)
  • Digested with HindIII and SpeI (Fast Digest Protocol, 20 ul reactions)
  • Ran on 1% agarose gel (protocol)
  • 
  • Cut out insert (top) bands for each reaction, keeping the two digests of each product in the same eppendorf tube.
  • Gel purified products (protocol)

6-17-2010

Ligated digested backbones and inserts. Transformed and plated on LB and Kanamycin.

• Prepared LB plates with 50 ug/ml kanamycin (15 ug of kanamycin per plate)
• Prepared 50 ug/ml LB+Kan Media.
• Ligated backbones and inserts of expression and reporter series vectors (ligation protocol)
  • Used 50 ng of digested, dephosphorylated backbone DNA
  • Used roughly 3x molar excess of digested vector
    • Mass of Insert = [Size of Insert][Molar excess][Mass of Vector]/[Size of Vector]
    • Size of Agrobacterium vector backbone: ~7 kb

Vector	Insert Size	Insert Mass
E3	600 bp	13 ng
E3	600 bp	13 ng
R1	2000 bp	20 ng
R3	1000 bp	40 ng

• Transformed ligation reactions into TURBO e. coli cells (protocol)
  • Transformed 5 ul of reaction mix into 15 ul of cells
  • Plated onto LB + Kan plates, left at 30ºC overnight

6-18-2010

Realized that there was a 1bp error in annealed oligos for O1 and O2 (had sticky end for SpeI site instead of NheI site)

• Ordered new oligos with corrected bp

Started to obtain E3, E4, R1 and R3 plasmids from E. coli

• Picked four colonies of E. coli from the E3, E4, R1 and R3 plates and made a culture from each in LB and Kanamycin
• Left cultures at 37ºC for 6 hours
• Centrifuged cultures to produce pellet and removed the liquid
• Stored pellet at -20ºC

6-21-2010

Carried out initial test to verify the identity of plasmid transformed into E. coli

• Carried out miniprep to obtain plasmids from cells (protocol link)
• Digested plasmids with EcoI and HindIII (protocol link)
• Ran digests on 1% agarose gel (protocol link)
• 
• Ladder was unclear so needed to repeat
• Measured concentration of miniprep product
  • Concentrations were low (<44ng/uL) so needed to redo miniprep
• Picked new colonies (2 from each plate) and grew cultures overnight

6-22-2010

Continued to carry out initial test to verify the identity of plasmid transformed into E. coli

• Carried out miniprep to obtain plasmids from cells (protocol link)
• Digested plasmids with EcoI and HindIII (protocol link)
• Ran digests on 1% agarose gel (protocol link)
• 
• Larger bands were observed, suggesting the correct backbones were present
• Smaller bands were observed, suggesting the correct inserts were present

Started to prepare for plasmids to be sequenced

• Required more plasmids
  • Transformed plasmids back into E. coli using heat shock (carried out twice for each of V9-12) (see protocol)
  • Added Kanamycin to LB plates
  • Spread E. coli cells onto LB + Kanamycin plates
  • Left plates in 37ºC overnight

6-23-2010

Continued to prepare for plasmids to be sequenced

• Picked 3 colonies from each of the V9, V10 and V12 plates and 4 colonies from each of the V11 plates
• Left at 37ºC for 6 hours
• After 6 hours, had not grown enough, so left cultures overnight at 37ºC

6-24-2010

Miniprepped V9-V12 cultures. Got yields with enough DNA to send to GeneWiz for sequencing. Primers arrived from Mr. Gene and we will send constructs off for sequencing next week.

Started putting together Open Series constructs

• Annealed oligos synthesized by Mr. Gene to form insert (protocol)
• Ligated with backbone digested last week
• Transformed into TURBO E. Coli and plated onto LB + Kan

6-25-2010

Continued preparation of Open Series constructs

• Picked colonies from both V7 (O1) and V8 (O2) and set up cultures. Only cultures for V7 grew. Because of the patterns of colonies on the plates, we suspect that the Kanamycin was not properly spread on the plates.
• We had used up all of our remaining digested backbone for O2, so we redid the SacII/SpeI digestion on both vectors with phosphatase, re-ligated, and re-transformed and plated, using a negative control

6-26-2010

Completed preparation of Open Series construct

• Colonies grew for V8 (O2) only. Set up cultures for colonies
• Miniprepped cultures of both constructs

6-28-2010

Prepared vectors for sequencing

• Resuspended primers in water
• Mixed required quantities and concentrations of each vector and primer in PCR tubes
• Sent tubes to GeneWiz for sequencing

6-30-2010

Got back sequencing results Media:Team_vector_sequence1.zip

• Confirmed V9, V10, V11, V12 - both #5,6 for all. V11 had a single base pair change in the middle of the GusA sequence (bp 6315, T instead of C). This basepair is at the end of a codon, and the change does not change the translation of the sequence.
• V7, V8 had faulty primers - the reverse was not the reverse compliment and the forward was too close to the sequence, so redesigned primers and ordered them.

7-1-2010

• Set up starter cultures for V9-V12 for maxipreps from colonies from the #5 plate of each construct (in LB+Kan 50ug/ml)
• Agrobacterium transformation (electroporation)
  • Transformed E4 and V10, plus did a control that was shocked without DNA, and a control that was not shocked but had E4 in it.
  • Plated on LB+Kan(50ug/ml)+Gent(30ug/ml)+Rif(10ug/ml)
• Poured started cultures in 150mL LB + Kan (50ug/ml)

7-2-2010

• Carried out maxipreps of V9-V12
• Created glycerol stocks of V9-V12

7-6-2010

• Agrobacterium plates had colonies for E4 and V10 and no growth for the negative controls
• Sequencing V7 and V8 showed they were correct
• Started cultures for maxiprep of V7 and V8