Difference between revisions of "Team:UGent Belgium/LabNotebook"

• We made a strong expression vector (pXS) by restriction of the linearized plasmid backbone pSB1C3 and insertion of gblock made up of promoter-RBS-cloning site-terminator-terminator (cut with EcoRI and PstI). Details
  • Details:

    Strong expression vector: pSB1C3-P_BBaJ23119-RBS_BBaB0034-GGsite_BsaI-TT_BBaB0010-TT_BBaB0012

    Restriction digests:

    1. Resuspend gBlocks in Elution Buffer (=TE) to concentration of 50 ng/uL -> 500 ng delivered => 10uL buffer
    2. Linearised PSB1C3: 25ng/uL at 50uL (used 2013 iGEM tubes)
    3. Prepare mixes (enzymes by NEB):
       
       

       Insert digest

       H2O	30	uL
       CutSmart buffer	10	uL
       EcoRI-HF	2	uL
       PstI	3	uL (tube 5 years past expiration date)
       gBlock 1	5	uL (final concentration 5 ng/uL)

       
       

       Backbone digest

       H2O	5	uL
       CutSmart buffer	10	uL
       EcoRI-HF	2	uL
       PstI	3	uL (tube 5 years past expiration date)
       linear pSB1C3	30	uL (final concentration 15 ng/uL)

       Incubate overnight in 37°C warm water bath (overdigestion because of enzyme expiration date and restriction sites close to fragment termini, especially for the gBlock).

• Our first attempts to isolate the inaZ gene from P. syringae and the inaX gene from X. campestris failed. Try inaZ with more stringent conditions and Primestar polymerase, perform more thorough lysis of X. campestris to try inaX again. Details
  • Details:

    The inaZ gene was sourced from BBa_K584027 using primers oM7628 and oM7629* (length 3.6 kbp, anneal at 58°C). The plasmid was obtained from KU Leuven (concentration unknown): PCR (Q5) ran with 10x and 1000x template dilutions, transformed into E. coli DH5a by heatshock, and plated on chloramphenicol.

    The inaX gene was sourced from X. campestris colonies using primers oM7626 and oM7627 (length 4.7 kbp, anneal at 70°C). Cloning overhangs were not included on these primers. The colony was suspended in 1000uL distilled water, vortexed, freezed 30 min. @ -80 deg. C, heated 20 min. @ 95 deg. C, vortexed, and debris spinned down.

  
  
  
  
  
  

• We transformed the strong expression vector (cf. 29/08) by electroshock into E. coli Top10, and plated it on chloramphenicol. Details
  • Details:

    Before transformation of the vector, we inactivated the restriction enzymes using heat: 20 min at 80°C, and did ligation without purification. We also purified the samples on a spin column, eluted in 10 uL.

    	length (bp)	conc. (ng/uL)	ratio	mass (ng)	volume (uL)
    Backbone	2070	15	1	50	3.33
    gBlock	263	5	3	19.06	3.81
    H2O	9.86	uL
    T4 ligase buffer	2	uL
    Backbone	3.33	uL
    Insert	3.81	uL
    T4 ligase	1	uL

• As a backup strategy we also used CPEC with the backbone from the K584027 plasmid (100x dilution) to generate a strong expression vector. For this, we used Primestar HS PCR with primers oM7643 and oM7645. Details
  • Details:

    Annealing: 5"@55C. Full program: 30x(10"@98C;5"@55C;2'@72C)

• Second attempt (cf. 29/08) to isolate the inaZ gene from P. syringae and the inaX gene from X. campestris. InaX failed, but inaZ was successful. Both Primestar HS and Primestar GXL PCR were used for this. On the gel, lane 4 and 8 are inaX attempts. Details
  • Details:

    For inaZ, we used BBa_K584027 as template (1/10, 1/100, and 1/1000 dilutions). Annealing temperature was 66.20°C for primer oM7628 and 61.93°C for primer oM7629* (length: 3.6 kbp). For inaX, we used X. campestris colonies as template. Annealing was done at 60.96°C for primer oM7626 and at 70.34°C for primer oM7627 (length 4.7 kbp).

    Conditions for Primestar HS: 1'@98C;30x(10"@98C;15"@60C;5'@68C);10'@68C (not the standard protocol).
    Conditions for Primestar GXL: 1'@98C;30x(10"@98C;15"@60C;5'@72C);10'@72C (not the standard protocol).

  
  
  
  
  
  
• Transformation of the strong expression vector by restriction and ligation was not very successful, we only had a few colonies. The backbone amplification using CPEC was successful (see gel, cf. 30/08). Details
  • Details:

    CPEC calculations for strong expression vector:

    	length (bp)	conc. (ng/uL)	ratio	mass (ng)	volume (uL)
    Backbone	2070	29	1	100	3.45	(diluted backbone 10x)
    Insert	263	5	3	38.12	7.62	(diluted gblock 10x)
    H2O	5.68	uL
    Q5 buffer	2	uL
    dNTPs (2mM)	5	uL
    Backbone	3.45	uL
    gBlock	7.62	uL
    DMSO	0.75	uL
    Q5 polymerase	0.5	uL

    Incubation: 30"@98C;15x(10"@98C;30"@55C;35"@72C);10'@72C.

  
  
  
  
  
  

• Heatshock transformation of the strong expression vector made with CPEC (cf. 31/08) yielded no colonies.
• Colony PCR on the colonies with the strong expression vector made with restriction and ligation didn’t show any positive ones (cf. 31/08). As primers, we used BioBrick verification primers (length: 534 bp).

Electroshock transformation of the strong expression vector made with CPEC (cf. 31/08) into E. coli Top10 cells.

• CPEC using Primestar GXL and an excess of insert. Details
  • Details:

    table5

• Via colony PCR we saw that all 14 colonies with the strong expression vector made with CPEC and electroshock transformation were all positive (cf. 02/09). We used BioBrick verification primers for this. Details
  • Details:

    Conditions: 2'@95C; 30x(20"@95C;30"@55C;1'@68C)

    table6

  
  
  
  
  
  
• We transformed E. coli Top10 cells with our weak expression vector (pXW), which came in today. Q5 CPEC was used for this with the linearized plasmid backbone pSB1C3, and as insert a gBlock consisting of promoter-RBS-cloning site-terminator-terminator. The promoter is weaker than the promoter of the strong expression vector (cf. 29/08). Details
  • Details:

    Incubation: 30"@98C;15x(10"@98C;30"@55C;35"@72C);10'@72C

    table7

Colonies 1 and 2 of our strong expression vector made with CPEC (cf. 04/09) are cultured and miniprepped. These are used for following constructs:

• pXS-INP_WT using the inaZ PCR fragment
• pXS-INP_NC-mGFPuv
• pXS-INP_NC-Strep (Strep: regular streptavidin)
• pXS-INP_NC-mSA2 (mSA2: monomeric streptavidin)
• pXS-Lpp-ompA-mGFPuv
• pXS-Lpp-ompA-Strep
• pXS-Lpp-ompA-mSA2

We transformed our Golden Gate reactions (cf. 06/08) into E. coli Top10 cells.

• We did a colony PCR of our weak expression vector (cf. 04/09), and of our constructs made with the strong expression vector (cf. 06/09), both using BioBrick verification primers. Details
  • Details:

    Expected length (bp) of the constructs made with the strong expression vector:

    • pXS-inaZ: 4115
    • pXS-INP_NC-mGFPuv: 2166
    • pXS-INP_NC-Strep: 1857
    • pXS-INP_NC-mSA2: 1832
    • pXS-lpp-ompA-mGFPuv: 1670
    • pXS-lpp-ompA-Strep: 1362
    • pXS-lpp-ompA-mSA2: 1338

    Conditions weak expression vector: 2'@95C; 30x(20"@95C;30"@55C;1'@68C)

    table11

• We tried to make inaZ Golden Gate safe by removing the internal BsaI sites. To achieve this, we used Primestar HS PCR and cut it into 3 different fragments. This was successful for the separate fragments, but we didn’t succeed to combine them. For the first fragment, this was simply from the original PCR fragment. The second fragment is a new PCR fragment, using oM7658 and oM7629 as primers and the inaZ plasmid as template (length:3039 bp). This band is smeared, probably due to excessive template concentration or annealing time. The third fragment is also a new PCR fragment, using oM7658 and oM7229 as primers and the inaZ plasmid as template (length: 293 bp).
  
  
  
  
  

The colony PCR of E. coli cells with the constructs with the strong expression vector showed no positive colonies (cf. 06/09). Not many bands are present, and all present bands show the original vector or self ligation. The colony PCR on our cells transformed with the weak expression vector (cf. 04/09) did show positive ones. We repeated the colony PCR under different conditions, but bands were still not of the correct lengths.
We therefore tried cloning all our parts into the weak expression vector by using a Golden Gate reaction mix. These Golden Gate reactions were then transformed into E. coli Top10 cells. Incubation was done at 30°C instead of 37°C to repress the plasmid copy number. Details

• We got our sequencing results: the strong expression vector is good!
• Colonies with the weak expression vector constructs are still too small to pick up with colony PCR (cf. 09/09).

• All our plates with the weak expression vector constructs show colonies! The control reaction has colonies as well, but much fewer, which was to be expected. Fluorescence is seen for the pXW-INP_NC-mGFPuv construct. All Lpp-ompA plates have a mixed phenotype, with 10-20 large colonies (same size as control reaction), and >60 small colonies (see culture picture). Colony PCR was performed using OneTaq at 50°C annealing temperature to proof that the small colonies are the positives ones, the larger ones the negative ones. This theory was confirmed for all colonies (see gel picture). Keep incubating at 30°C to reduce copy number.
  
  
  

  
  
  
• We performed Primestar HS PCR to create following constructs:
  • pXW-Lpp-ompA-mGFPuv-(m)Strep using Lpp-ompA-mGFPuv fragment. As a template, we used the Golden Gate mix for pXW-Lpp-ompA-mGFPuv, and oMEMO7667_Lpp_Fw and oMEMO7663_GFP_Rv-ATCT as primers (expected length:1167).
  • pXW-mGFPuv-m(Strep) using mGFP. As a template, we used a 100x diluted gBlock, and oMEMO7662_GFP_Fw-GATG and oMEMO7663_GFP_Rv-ATCT as primers (expected length: 752 bp).
  • pXS-mGFPuv using mGFP. As a template, we used a 100x diluted gBlock, and oMEMO7662_GFP_Fw-GATG and oMEMO7664_GFP_Rv-TACT as primers (expected length: 752 bp).
  We also repeated previous PCR (cf. 08/09) of the largest piece of inaZ to make it Golden Gate safe. As a template, we used the inaZ plasmid, and oM7658 and oM7629 as primers (expected length: 3039 bp). Because the band was smeared last time, we should either use lower template concentrations or a shorter anneal time.

• Miniprep of a number of colony PCR positive colonies (cf. 09/09).
• Next picture is the gel of the PCR reactions of the previous day (cf. 11/09). inaZ was run 2 times (at 1/100 and 1/1000 dilution), these are the first 2 bands. The next 3 bands are the 3 constructs. PCR using the Golden Gate mix as a template didn’t work very well (two extra bands, lane 3). We therefore repeated this reaction using a pure plasmid.
  
  
  
  
  
• Golden Gate assemblies of:
  • pXW-inaZ_GGsafe
  • pXW-mGFPuv-Strep
  • pXW-mGFPuv-mSA2
  • pXW-mGFPuv
  • pXS-mGFPuv
  Details
  • Details:

    table13

• pXW-Lpp-ompA-mGFPuv miniprep tubes that did not grow yesterday (cf. 12/09) did show growth today, probably through mutations.
• Transformed Golden Gate assemblies (cf. 12/09) into E. coli Top10 cells.

Colony PCR of the transformed Golden Gate assemblies (cf. 13/09): fail for pXW-INP_GGsafe (first 7 lanes), okay for other assemblies. For pXW-INP_GGsafe, we will make a new Golden Gate design.

Colony PCR screening of 8 more colonies with pXW-inaZ_GGsafe showed no result (cf. 13/09)

• Minipreps of the following plasmids and sequencing with verification primers:
  • pXW-mGFPuv-Strep
  • pXW-mGFPuv-mSA2
  • pXW-mGFPuv
  • pXS-mGFPuv
• We did a Primestar HS PCR to go from high copy to low copy (LC) backbone, with as backbone pSB3K3 and 4 inserts: INP_NC-Strep, INP_NC-mSA2, Lpp-ompA-Strep and Lpp-ompA-mSA2. The backbone and Lpp-ompA-mSA2 both failed. On the gel, all four inserts are seen. The backbone was run on a different gel (not pictured here). Details
  • Details:

    Backbone: pSB3K3 with pSB3K3 as template (plate4, F6, iGem distribution 2016), and oMEMO7669 & oMEMO 7670 as primers (length: 2790 bp).
    Insert1: INP_NC-Strep (156 ng/ul) with pXW-INP_NC-Strep as template, and oMEMO7671 & oMEMO 7672 as primers (length: 1684 bp).
    Insert2: INP_NC-mSA2 (290 ng/ul) with pXW-INP_NC-mSA2 as template and oMEMO7671 & oMEMO 7672 as primers.
    Insert3: Lpp-ompA-Strep (130 ng/ul) with pXW-Lpp-ompA-Strep as template, and oMEMO7671 & oMEMO 7672 as primers.
    Insert4: Lpp-ompA-mSA2 with pXW-Lpp-ompA-mSA2 as template, and oMEMO7671 & oMEMO 7672 as primers.

  
  
  
  
  
  

• We retried the Primestar HS PCR to go from high to low copy backbone. The backbone failed again, Lpp-ompA-mSA2 was successful. We also made the ‘new parts’ of inaZ, was was also successful. Details
  • Details:

    Retry backbone: used pSB3K3 as template, and oMEMO7675 & oMEMO7644 as new primers (length: 2790 bp).
    Retry insert4 (Lpp-ompA-mSA2): used Golden Gate mix as template (134 ng/ul), and oMEMO7671 & oMEMO7672 as primers.
    New parts inaZ: inaZ plasmid as template, and oMEMO7628 & oMEMO7673 as primers for piece1 (150 ng/ul, 634 bp), oMEMO7658 & oMEMO7674 as primers for piece2 (146 ng/ul, 2792 bp), and oMEMO7659 & oMEMO7629 as primers for piece3 (122 ng/ul, 297 bp).

  
  
  
  
  
  
• Golden gate assembly of pXW-inaZ_GGsafe. Details
  • Details:

    table15

• We did a colony PCR for the Golden Gate assembly of pXW-inaZ_GGsafe (cf. 19/09). Colony 2, 6, 8, and 32 are preculture. 13 also kind off looks okay, but is too small in length (probably due to some failed fragments).
  
  
  
  
  
• We opted for a new backbone for the low copy vector (cf. 19/09): pSC101-Kan vector.

• We digested colony PCR samples 2, 6, 8, 13, and 32 (cf. 21/09), and a control with BsaI. If pXW-inaZ_GGsafe is indeed Golden Gate safe, it should not be cut. This was okay! Colony 2 and 6 are plasmid prepped for sequencing. Colony 13 is lane 5, control is lane 6.
  
  
  
  
  
• We again retried the Primestar HS PCR to go from high to low copy backbone. For this, we have the low copy backbone pSC101 (right gel, lane 1). A new Lpp-ompA-mSA2 insert was also done to obtain an insert for the low copy vector (left gel, lane 1). A weak expression vector with an empty expression site was also added (left gel, lane 2), and also inaZ-RC. Lane 2 and 3 on the right gel are respectively a control, and backbone pSB1C3 (mSA2-REsafe gBlock assembly). Details
  • Details:

    New Backbone "LC" (pSC101): pSC101-Kan vector as template, and oMEMO7714 & oMEMO7715 as primers (length: 4034 bp).
    New Lpp-ompA-Strep insert: Golden Gate mix as template, and oMEMO7671 & oMEMO 7672 as primers (1051 bp).
    XW empty expression site: pXW as template, and oMEMO7671 & oMEMO7672 as primers (length: 224 bp).
    inaZ-RC: pXW-inaZ_GGsafe as template, and oMEMO7665 & oMEMO7666 as primers (length: 3100 bp).

  
  
  
  

  
  
  

CPEC assemblies to create:

• pLC-XW
• pLC-XW-INP_NC-Strep
• pLC-XW-INP_NC-mSA2
• pLC-XW-Lpp-ompA-Strep
• pLC-XW-Lpp-ompA-mSA2
• pSB1C3-mSA2

Colony PCR of all CPEC assemblies (cf. 26/09) with primers oMEMO3441 + oMEMO1760 for the first 5 assemblies and verification primers for the last assembly. The order of the gel is: pLC-XW-INP_NC-Strep, pLC-XW-INP_NC-mSA2, pLC-XW-Lpp-ompA-Strep, pLC-XW-Lpp-ompA-mSA2, pSB1C3-mSA2, and pLC-XW (only 3 colonies).

• Second attempt for CPEC assemblies (cf. 26/09), with increased backbone DNA (200 ng) and an altered backbone/insert ratio (1:5). Details
  • Details:

    table17

• Primestar HS PCR to create GATG-mGFPuv-ATCT, which is necessary for new attempts for the pXW-mGFPuv-Strep and pXW-mGFPuv-mSA2 constructs, due to mutations in previous versions. For this, we used primers oMEMO7662 + oMEMO7663, and pXS-mGFPuv as template.
  
  
  
  
  
• Golden Gate assemblies to create:
  • pXW-inaZ-RC+Strep
  • pXW-inaZ-RC+mSA2
  • pXW-inaZ-RC+GFP
  • pXW-mGFPuv-Strep
  • pXS-mGFPuv-Strep
  • pXS-mGFPuv-mSA2
  Details
  • Details:

    table18

    table19

• We also started cultures for the cell binding assay to test the attachment to our filament, and for protein extraction. For the first, we used constructs pXW-INP_NC-Strep + pSC101-RFP, pXW-INP_NC-mSA2 + pSC101-RFP, and pSC101-RFP. For the latter, we used constructs pXW-mGFPuv-mSA2 and pXW-mGFPuv.

• Sequenced two new colonies of construct pXW-mGFPuv-Strep (due to mutation in GFP).
• Created pSB1C3-mGFPuv2 for submission to the parts registry. For this we amplified the backbone from pSB1C3 linear template using primers oM7722 and oM772 (length: 2093 bp), and mGFPuv2 from construct pXW-mGFPuv or pXS-mGFPuv using primers oM7662 and oM7664 (length: 744 bp). Golden Gate assembly was done using BsaI.

We did colony PCR on the CPEC assemblies with increased backbone DNA and altered backbone/insert ratio (cf. 28/09), and previous Golden Gate assemblies (cf. 28/09). We found positive colonies for following constructs:

• pLC-XW
• pLC-XW-INP_NC-Strep
• pLC-XW-INP_NC-mSA2
• pXW-inaZ-RC+mSA2
• pXW-mGFPuv-Strep
• pXS-mGFPuv-Strep
• pXS-mGFPuv-mSA2

New colony PCR of 2x14 colonies of constructs pXW-inaZ-RC+Strep and pXW-inaZ-RC+GFP.

We found a positive colony for construct pXW-inaZ-RC+GFP (cf. 03/10).

Golden Gate assembly of pSB1C3-mGFPuv2, with a shorter protocol (cf. 29/09), and transformation into E. coli Top10 cells. Details

• Colony PCR on 14 pSB1C3-mGFPuv2 colonies (cf. 05/10) (plates almost overgrown, assembly was apparently very efficient). For primers, we used verification primers (length: 1030 bp).
  
  
  
  
  
• Combined pXW-INP_GGsafe with pLC-XW-INP_NC-mSA2 by electroporation (plate on kan+chloramphenicol).

Sequencing results for constructs 13-24: