Lab Notebook - Jenn

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June 2009

Monday, June 22

First Round of 2AB assembly of 140L composite parts is almost complete. I plated cells on friday (see excel sheet "Jenn 140L Guide/ManualMiniRobotTest" for locations of parts on LB agar strips) and picked colonies today.
* This round of assembly was done with manual minipreps of plasmids from cells in the "140L neg80 stock plate" because the MacConnell Research Miniprep96 proved to be unreliable.
-The colonies were spot checked manually on AKC and dual antibiotic plates to check for efficacy of the assembly.
-LB Agar strip locations P2:A1-A4 yielded VERY small colonies.
The next round of assembly will be done with the excel sheet "Jenn 140L Guide/Next Round").

Note for future assemblies: be careful with how much LB agar is poured into each strip.

Tuesday, June 23

Miniprepped colonies from LB Agar Agar Plate "140L Jenn 6-23-09" by hand. All miniprepped plasmids were named according to synthbiobootcamp google doc and put in "Jenn's 140L" -20 box.
Found parts tim and gabe made from 140L and transformed some plasmids into Righy because there was not very much DNA left.

Wednesday, June 24

Started assembly of 140L goal parts. Did P4 plate (see Jenn 140L guide for reference). P4 should assemble 96 things, M10400-M10567. Nterm displayers and Cterminal displayers were grouped separately on LB agar strips to make pouring agar easier.

I cannot remember if i added ligase to the ligase cocktail, this round of assembly may not have worked :(

Thursday, June 25

Re-did assembly of the 140L goal parts (P4) because i cant remember if i added ligase. I got some very small colonies in the morning and most grew to normal looking colonies in the afternoon (the KC colonies stayed very small, those strips were poured with a different LB agar and may have been bad), but i had already started the assembly again. This round of assembly is labeled as P4#2.
We are suspicious of the new stocks of DH10B cells (both my plates and the iGEM plates have very small colonies that look like contamination. We are spot testing the DH10B cells to see if they are contaminated. In the mean time iGEM and 140L assembly set will be done with TG1 cells.
The gels for the second round (P4#2) look better than last time.

Friday, June 26

Colonies from the P4#2 look very strange. Some strips are lawns, some are very large with spread edges (they are not round like usual colonies). I decided to pick colonies today into 96 well culture dishes. The LB+antibiotic was mixed with 50% glycerol to a final concentration of 8% glycerol.

Sunday, June 28

Spot checked the P4#2 glycerol stock plates onto AKC and C to see if parts are correct/co-transformed. The glycerol stock plate looks questionable (i'm not sure if things grew in every well). I'd like to pick colonies from P4#1 into glycerol stock plates, but i dont have time today.
I also re-picked pBca9495KC-M10318 and pBca9495KC-M10319 from the 140L LB agar strips because the last time there were no correct colonies picked. I need these constructs to do the next (P6) set of 140L goal parts.

Monday, June 29

Miniprepped pBca9495KC-M10318 and pBca9495KC-M10319 and added them to the "M10300...miniprep" plate of frozen un-diluted miniprep to do assembly tomorrow. Also re-organized the excel spreadsheet for the 140L goal parts to prep them for assembly tomorrow.
Twelve colonies came back co-transformed on both AKC spot test plates (this is for the P4#2 140L parts). The co-transformed parts were located at B12, C4, C12, D7, D9, D11, F4, F10, F12, G3, G10, and H2. New colonies of these constructs were picked and spotted on AKC. If a colony of each construct comes back correctly tomorrow the first plate of 140L goal parts should be assembled.

  • We will not be miniprepping all 96 goal parts from P4 (because thats to many things to miniprep by hand). But I am going to miniprep these things tomorrow to check for correct assembly with restriction mapping (EcoRI/BamHI): A2 (M10401)(4259/2974), B4 (M10455)(3653/2974), D10 (M10421)(3039/1848), E6 (M10433)(3039/1917), F8 (M10481)(3315/3039), G4 (M10493)(3039/2823)

Tuesday, June 30

Miniprepped A2 (M10401), B4 (M10455), D10 (M10421), E6 (M10433), F8 (M10481), G4 (M10493)

  • Did restriction digest with EcoRI/BamHI:
  • ladder, A2 (M10401)(2974/4259), B4 (M10455)(2974/3653), D10 (M10421)(3039/1848), E6 (M10433)(3039/1917), F8 (M10481)(3039/3315), G4 (M10493)(3039/2823)


  • Did another restriction map with less DNA (0.5uL), same expected band sizes:


  • Sent A2 (JAB028/JAB029), D10 (JAB030/JAB031), and F8 (JAB032/JAB033) in for sequencing with ca56/G00101.

Ten samples (B12, C4, C12, D7, F4, F10, F12, G3, G10, and H2) came back good on the spot tests. These were added to the "P4 140L goal" stock plate.

  • Five new colonies of D9 and D11 constructs were picked and spotted on AKC. If a colony of each construct comes back correctly tomorrow they will be added to the "P4 140L Goal" stock plate.

Transformed strains for the ELISA strep assay. Transformed pBca9145-9494(+ control), pBca9523-1363(- control), and pBca9495CA-1144#5 (fluorescent) into TG1, DH10B, and MC1061. Colonies were innoculated overnight in LB with the correct antibiotics. A test of cell adhesion to the surface of maxisorp plates will be done tomorrow. Three strains were picked to see if cell type affected the ability to adhere to the plate.

July 2009

Wednesday, July 1

Sequencing of JAB028-JAB033 came back very strange. Every part was missing its passenger, and two of the three displayers were mixed up.
I decided to restriction map the minipreps from "140L manual minipreps stock plate" to see if the parts that we thought were in Righty/Lefty were indeed in the correct cell lines. Below is the gel (BamHI/XhoI digest) here are the lanes/expected band sizes:
1. ladder
2. M10043(lefty:1232/1975)
3. M10053(lefty:1316/1975)
4. M10033(lefty:1247/1975)
5. M10087(lefty:1508/1975)
6. M10043(righty:3207)
7. M10053(righty:3291)
8. M10033(righty:3222)
9. M10087(righty:3483)
The gel shows that the expected lefty parts are mono-cut and therefore righty, and the expected righty parts have multiple bands and are therefore not righty. It is unclear what the last four lanes on the gel hold. Lanes 6 and 7 do not have the correct size bands, even if they are lefty.
I ran another restriction map (this time of the parts directly from their miniprep tubes (although i suspect that they were messed up on the original 140L plate/spreadsheet. Below is the gel (BamHI/XhoI digest), the lanes/expected band sizes are the same as the previous gel:
1. ladder
2. M10043(lefty:1232/1975)
3. M10053(lefty:1316/1975)
4. M10033(lefty:1247/1975)
5. M10087(lefty:1508/1975)
6. M10043(righty:3207)
7. M10053(righty:3291)
8. M10033(righty:3222)
9. M10087(righty:3483)
Decided to innoculate colonies from the -80 140L stock plate to see if problem originated there.

Thursday, July 2

Yesterday I picked M10043 and M10053 colonies from both the designated Righty and Lefty positions in the -80 140L stock plate to see where the righty/lefty switch occured. I miniprepped those colonies today, and restriction mapped them. Unfortunately the restriction map (BamHI/XhoI) was inconclusive:
1. ladder
2. M10043(lefty:1232/1975)
3. M10053(lefty:1316/1975)
4. M10043(righty:3207)
5. M10053(righty:3291)

  • Maybe my minipreps were bad. I will pick the colonies again and try this again tomorrow.

Today i transformed some of the relevant 140L parts into TG1 cells to grow up more DNA for an EcoRI/BamHI transfer into pBca9523 entry vectors.
1. <KILR> pBca9495AK-M10037
2. <EILD> pBca9495AK-M10053
3. <CBK> pBca9495AK-M10046
4. <Z34C domain> pBca9495AK-M10018
5. <Beta Roll> pBca9495AK-M10022
6. <Beta Helix> pBca9495AK-M10089
7. <GFP-LVA> pBca9495AK-M10025
8. <INP repeats> pBca9495AK-M10087
We also had our first mini-meeting today, and i will soon begin to design composite parts.

Friday, July 3

Re-did neg80 test:
1. ladder
2. M10043(lefty:1232/1975)
3. M10053(lefty:1316/1975)
4. M10043(righty:3207)
5. M10053(righty:3291)
Now we can tell that the neg80 stock plate was correct and i messed up the lefty/righty strains.
Miniprepped <KILR> pBca9495AK-M10037, <EILD> pBca9495AK-M10053, <CBK> pBca9495AK-M10046, <Z34C domain> pBca9495AK-M10018, <Beta Roll> pBca9495AK-M10022, <Beta Helix> pBca9495AK-M10089, <GFP-LVA> pBca9495AK-M10025, and <INP repeats> pBca9495AK-M10087.

  • Digested pBca9495-1144#5 and gel purified out the vector.
  • Did Eco/Bam transfer of the passengers into pBca9523 entry vector. Plated on spec plates.

Made an iGEM09 Parts google doc because clotho is not finished enough to be useful. Main complaint= cant sort, search, or delete things in the parts manager.

Monday, July 6

Picked colonies of pBca9523-(<KILR> M10037, <EILD> M10053, <CBK>M10046, <Z34C domain> M10018, <Beta Roll> M10022, <Beta Helix> M10089, <GFP-LVA> M10025, and <INP repeats> M10087).

  • Will miniprep and restriction map them tomorrow

Transformed the rest of the relevant 140L parts into TG1 cells to grow up more DNA for an EcoRI/BamHI transfer into pBca9523 entry vectors.
1. (dblterm) pBca9495CA-Bca1092
2. (Pbad.rbs.prepro.streptag) pBca9495CA-Bca1363
3. (AraC-Pbad2)(rbs1179) pBca9495KC-Bca9476
4. (<streptag!)(dblterm) pBca9495KC-9496
5. (<GS5-IILK>) pBca9495AK-M10049

Tuesday, July 7

Miniprepped pBca9495CA-Bca1092 pBca9495CA-Bca1363, pBca9495KC-Bca9476, pBca9495KC-9496, and pBca9495AK-M10049. Then Eco/Bam transferred them into pBca9523.
Designed composite parts.
Had Joey miniprep pBca9523-M10037, -M10053, -M10046, -M10018, -M10022, -M10089, -M10025, and -M10087. Wanted to restriction map them, but we ran out of gels. Will try tomorrow. Also re-innoculated the bacteria just in case.

Wednesday, July 8

Did a restriction map (EcoRI/BamHI) of pBca9523-M10037, -M10053, -M10046, -M10018, -M10022, -M10089, -M10025, and -M10087 that Joey miniprepped yesterday. Expected band sizes:
1. pBca9523-M10022 (411/2472)
2. pBca9523-M10046 (135/2472)
3. pBca9523-M10087 (303/2472)
4. pBca9523-M10089 (678/2472)
5. pBca9523-M10018 (108/2472)
6. pBca9523-M10025 (756/2472)
7. pBca9523-M10037 (111/2472)
8. pBca9523-M10053 (111/2472)

  • Everything looks good except for lane 7 (M10037). So i miniprepped that part again.

Miniprepped pBca9523-Bca1092 -Bca1363, -Bca9476, -9496, and -M10049, and restriction mapped them (EcoRI/BamHI). Expected band sizes:
1. pBca9523-Bca1092 (135/2472)
2. pBca9523-Bca9476 (1306/2472)
3. pBca9523-M10049 (147/2472)
4. pBca9523-Bca9496 (207/2472)
5. pBca9523-Bca1363 (1458/2472)
6. pBca9523-M10037 (111/2472)
Everything looks good but the enzymes i chose may not have resolved the plasmids well enough. So did a new restriction map with BglII/EcoRV:
1. pBca9523-M10037 (326/2266) UNWANTED (3291)
2. pBca9523-M10053 (326/2266) UNWANTED (3291)
3. pBca9523-M10046 (350/2266) UNWANTED (3315)
4. pBca9523-M10018 (323/2266) UNWANTED (3288)
5. pBca9523-M10022 (69/557/2266) UNWANTED (69/3522)
6. pBca9523-M10089 (893/2266) UNWANTED (3858)
7. pBca9523-M10025 (971/2266) UNWANTED (3936)
8. pBca9523-M10087 (518/2266) UNWANTED (3483)
next gel
1. pBca9523-Bca1092 (350/2266) UNWANTED (892/2291)
2. pBca9523-Bca9476 (338/1183/2266) UNWANTED (338/1300/2651)
3. pBca9523-M10049 (362/2266) UNWANTED (3327)
4. pBca9523-Bca9496 (422/2266) UNWANTED (1890/1300)
5. pBca9523-Bca1363 (338/1335/2266) UNWANTED (338/892/3276)
Gateway3.jpg Ran gels longer--> Gateway4.jpg Gateway5.jpg

Thursday, July 9

Looked at spot checks of pBca9523? parts on spec and their previous vectors' plates. Decided that Bca1092, Bca1363, M10049, Bca9496, and Bca9476 are definitely in pBca9523. The other constructs are a little more ambiguous so i miniprepped other clones of those samples today. Here is a restriction digest of these eight parts (XhoI/AlwNI):
1. pBca9523-M10037 (320/2272) UNWANTED (1381/1910)
2. pBca9523-M10053 (320/2272) UNWANTED (1381/1910)
3. pBca9523-M10046 (320/2296) UNWANTED (1381/1934)
4. pBca9523-M10018 (320/2269) UNWANTED (1381/1907)
5. pBca9523-M10022 (320/2572) UNWANTED (1381/2210)
6. pBca9523-M10089 (320/2839) UNWANTED (1381/2477)
7. pBca9523-M10025 (320/1351/1566) UNWANTED (674/1381/1881)
8. pBca9523-M10087 (320/2462) UNWANTED (1381/2102)
Tested out the 8-strip Minipreps today. Added all of the correct buffers. To pellet cells: spin 5500g for 5 min. To pellet cell debris: spin 5500g for 7 min. All other spins: 2500g for 2 min. Gaby ran a restriction map of the resultant DNA (EcoRI/BamHI):
1. pBca9523-9496 (207/2472)
2. pBca9523-9496 (207/2472)
3. pBca9523-M10049 (147/2472)
4. pBca9523-M10049 (147/2472)
5. ladder 6. pBca9523-Bca9476 (1306/2472)
7. pBca9523-Bca9476 (1306/2472)
8. pBca9523-Bca1092 (135/2472)
9. pBca9523-Bca1092 (135/2472)
The minipreps look good, and like they recover a good amount of DNA. Also began to gateway basic parts into assembly vectors using in vivo gateway. I followed the in vivo gateway protocol that Madhvi gave me, and grew everything in LB+spec in 24 well blocks overnight.

Friday, July 10

Miniprepped assembly vectors from the pir-gateway cells using the 8-strip minipreps. The supernatant of cell lysate was very hard to aspirate. Gaby restriction mapped 8 of the minipreps. Here are the expected band sizes (EcoRI/BamHI):
1. pBdr052AK-B09ig314 (2541/9141)
2. pBdr052AK-B09ig171 (816/9141)
3. pBdr052AK-M10087 (303/9141)
4. pBdr052KC-B09ig1 (834/8649)
5. ladder 6. pBdr052KC-B09ig278 (864/8649)
7. pBdr052KC-B09ig310 (1425/8649)
8. pBdr052AK-B09ig326 (327/9141)
9. pBdr052AK M10018 (108/9141)
Re-transformed them into pir-Righty and pir-Lefty cells, then plated them on LB agar strips.

Saturday, July 11

Took the strips out of the incubator.

Sunday, July 12

The cells did not grow evenly on the strips. There were some strips that had a lot of colonies, and there were others that had few to no colonies. I re-transformed all of the parts that had few to no colonies into pir-Righty and pir-Lefty cells: (strip# A1)B09ig314, (A2)B09ig318, (A3)B09ig322, (A5)B09ig326, (A7)B09ig239, (A8)B09ig145, (A12)M10046, (B3)M10089, (B4)M10025, (D8)Bca1092.

Monday, July 13

Didnt see much improved growth for the constructs that i retransformed yesterday, so we are going to start those over from the gateway transformation step. When re-transforming the basic parts we labeled them according to the strips that they were supposed to be plated on (at the step that failed).

  • We also went into liquid culture at the gateway cell transformation that had both spec and the assembly vectors antibiotic (ie-spec & AK) so that we were selecting for cells that kept the assembly vector.

We also re-spot checked the clones that Gaby picked yesterday, this time using a pin tool. A few of the pins picked up ALOT of cells (plate1-C5,C11,C12,D4, Plate2-C11?, Plate3-C1?D1?)

Tuesday, July 14

Miniprepped all of the basicAssemblyRL1 parts that were not co-transformed. Restriction mapped a few of these parts that should be in assembly vectors (BamHI/XhoI):
1. ladder
2. B09ig324{<SOD>}-RIGHTY (9609)
3. B09ig328{<cel6A!}-RIGHTY (11157)
4. B09ig239{<mgfp-5>}-RIGHTY (9375)
5. M10046{<CBK>}-RIGHTY (9276)
6. M10089{<Beta Helix>}-RIGHTY (9819)
7. Bca1363{Pbad.rbs.prepro.StrepTag}-LEFTY (7620/2487)
8. B09ig306 {<yuaQ AtD>}-LEFTY (7450/2072)
9. Bca1092(dblterm)-RIGHTY

  • The gel looked okay, but not very informative. we began to do assembly anyway.
  • Plated in 1 12x2 set of strips and 2 KA plates.

Gaby miniprepped the 8 things that did not gateway properly the first time around (LB-strip# A1)B09ig314 <cel3A>, (A2)B09ig318 <cel5B>, (A3)B09ig322 <cel9A>, (A5)B09ig325 <TirM>, (A7)B09ig239, (A8)B09ig145 <needle scFv>, (B4)M10025 <GFP-LVA>, (B6), (D8)Bca1092). She restriction mapped them (see her notebook) and they looked good. The assembly vectors showed much stronger bands this time. She then transformed them into pir-RIGHTY cells and plated them.

Wednesday, July 15

Picked colonies from yesterday's assembly. Did 4 replicates of each, grew them up in 1mL of 2YT+ the correct antibiotics. ___hours later spot checked them onto big AKC plates so that tomorrow we can know about co-transformation and will be able to miniprep all of the successful parts.

  • Tomorrow i will miniprep the clones that were not co-transformed and will restriction map a few and send them in for sequencing
  • The colonies with <pbad.rbs.prepro.streptag><passenger> parts were very small this morning. Is this because the cells were over-crowded? Or is this because the parts are toxic?

There were 3 assemblies that didnt work (they had no colonies grow): C09ig0027{<VtaA11 AtD>}(dblterm), C09ig0035{<ehaB>}(dblterm), and C09ig0038{<VirG AtD>}(dblterm). Susan did 2ab assembly with them today to get them to grow.

  • note- very few things grew in this assembly, they were not analyzed because they were transformed into the wrong strain (Lefty) and the other parts came back strange.

Thursday, July 16

All <pbad.rbs.prepro.streptag><passenger> parts assembled on tuesday were co-transformed. I thought this might be due to a faulty <pbad.rbs.prepro.streptag> (Bca1363) part, so i restriction mapped it. This should be pBdr052CA-Bca1363 in Lefty so i digested with:
1. BamHI/XhoI (7620/2564)
2. BglII/XhoI (big)

  • The gel came back very strange. There were two very large bands suggesting that the plasmid was only single cut. Chris thinks this may be due to a mutated ccdB gene in the assembly vector which snuck in during gateway. Five more clones of pBdr052CA-Bca1362 LEFTY were picked today and will be spot checked and restriction mapped so that we may assemble things tomorrow. We also began to re-gateway this part into pBdr052CA.

I also wanted to test the passengers of the <pbad.rbs.prepro.streptag><passenger> parts that all failed to be sure that the failure was due to a faulty Bca1363, and not faulty passengers. Here is the restriction map digested with BglII/XhoI(all parts were RIGHTY):
1. B09ig324{<SOD>}(2955/6654)
2. B09ig328{<cel6A!}(4503/6654)
3. B09ig239{<mgfp-5>}(2721/6654)
4. B09ig171{<gliadin binding scFv>}(3303/6654)
5. M10037{<KILR>}(2598/6654)
digested with BglII/XhoI:

There were 3 other parts that failed in the assembly from tuesday: C09ig0027{<VtaA11 AtD>}(dblterm), C09ig0035{<ehaB>}(dblterm) C09ig0038{<VirG AtD>}(dblterm). We decided to check the basic parts to see if there were problems with those basic parts:
1. ladder
2. <VtaA11 AtD> Lefty(BamHI/XhoI)(6820/2072)
3. <ehaB>(BamHI/XhoI)Lefty (2072/7807)
4. <virG>(BamHI/XhoI)Lefty (2072/8008)
5. <dblterm>(BglII/XhoI)Righty (2699/6162)

  • Can see that the autotransporters were probably also ccdB mutants. Four more clones of pBdr052CA-Bca1362 LEFTY were picked today and will be spot checked and restriction mapped so that we may assemble things tomorrow. We also began to re-gateway these parts into pBdr052KC.

After finding out that the spot checking may not be enough of a screen for gateway products, we decided to restriction map a few of the most important parts that are supposed to be in assembly vectors just to make sure they were correct. Here is a restriction map of the passengers in Lefty (BamHI/XhoI digest) pBdr052AK-
1. B09ig201{<Cub>}(6300/2487)
2. B09ig207{<Nub>}(6309/2487)
3. B09ig213{<caspace 3>}(6996/2487)
4. B09ig249{<Tev N>}(6522/2487)
5. B09ig261{<Tev C>}(6540/2487)
6. pBdr052CK-Bca1363{Pbad.rbs.prepro.StrepTag}(7620/2487)

  • These look good, so they will be okay to use for assembly.

Miniprepped a bunch of things (newly gateayed parts and seemingly successful composite parts) using the 8-well strips. These things were restriction mapped:
1. B09ig314 {<cel3A!}pBdr052AK (5028/6654) (BglII/XhoI)
2. B09ig145{<type IIIs Needle Complex scFV>}pBdr052AK (3324/6654)(BglII/XhoI)
3. Bca1092(dblterm)pBdr052KA (2699/6577) (BglII/XhoI)
4. Bca1092(dblterm)pBdr052KA (2699/6577) (BglII/XhoI)
5. C09ig0028{<Hag AtD>}(dblterm)singlecut (BamHI/XhoI) UNWANTED (6955/2564)
6. C09ig0040{<AIDA-1 AtD>}(dblterm)singlecut(BamHI/XhoI)UNWANTED (8137/2564)
7. C09ig0028{<Hag AtD>}(dblterm)(2942/6577) (BglII/XhoI)
8. C09ig0040{<AIDA-1 AtD>}(dblterm) (4124/6577) (BglII/XhoI)

  • 1-4 look good and can be used for assembly
  • 5-8 are all ambiguous

Ran another restriction digest of the <displayer><term> parts that were miniprepped today to see if they were correctly righty methylated (and to clear up the ambiguity from the last gel).
1. ladder
next four are bamHI/XhoI digested:
2. pBdr052KA-C09ig0024{<azo1653 AtD>}(dblterm) (1000ish)
3. pBdr052KA-C09ig0028 {<Hag AtD>}(dblterm)
4. pBdr052KA-C09ig0031 {<upaG_short>}(dblterm) (1000ish)
5. pBdr052KA-C09ig0040 {<AIDA-1 AtD>}(dblterm) (10701)
next four are bglII/XhoI digested:
2. pBdr052KA-C09ig0024{<azo1653 AtD>}(dblterm) (3533/6577)
3. pBdr052KA-C09ig0028 {<Hag AtD>}(dblterm) (2942/6577)
4. pBdr052KA-C09ig0031 {<upaG_short>}(dblterm) (2969/6577)
5. pBdr052KA-C09ig0040 {<AIDA-1 AtD>}(dblterm) (4124/6577)

  • It looks like all of the vectors are missing their parts.

Decided to test the basic parts from BasicAssembleRL1.
1. pBdr0052KC-B09ig1{<azo1653 AtD>} (BamHI/XhoI)Lefty (7411/2072)
2. pBdr052KC-B09ig121{<Hag AtD>} (BamHI/XhoI)Lefty(6820/2072)
3. pBdr052KC-B09ig277{<upaG_short>} (BamHI/XhoI)Lefty(6847/2072)
4. pBdr052KC-B09ig310{<AIDA-1 AtD>} (BamHI/XhoI)Lefty(8002/2072)
5. <dblterm>(BglII/XhoI)Righty (2699/6162)

Friday, July 17

Chris thinks that the composite parts may have been put into Lefty instead of Righty (which may be true). We miniprepped new clones and ran restriction maps to see if he was correct.

  • They look exactly the same as the first gels we ran

We also miniprepped new clones of the basic parts that had trouble gateway-ing: pBdr052CA-Bca1363 in Lefty, <VtaA11 AtD> Lefty(BamHI/XhoI), <ehaB>(BamHI/XhoI)Lefty, <virG>(BamHI/XhoI)Lefty to see if we could get good clones without re-gateway-ing.
1. <Bca1363> BamHI/XhoI (7620/2564)
2. <VtaA11 AtD> Lefty(BamHI/XhoI)(6820/2072)
3. <ehaB>(BamHI/XhoI)Lefty (2072/7807)
4. <virG>(BamHI/XhoI)Lefty (2072/8008)
Anothergatewaytest.jpg colony PCR of this Bca1363 (expected band size: 1458)ColonyPRCpadpbad.jpg

  • The colony PCR of Bca1363 looks bad, the restriction map of everything except maybe <VtaA11 AtD> looks bad.

Tried colony PCR of another clone of <Pbad...>, <VtaA11>, <EhaB> and <virG>. Here are the expected band sizes (ca998/G00101):
1. ladder
2. Bca1363 (1458)
3. <VtaA11 Atd> B09ig113 (243)
4. <EhaB> B09ig281 (1230)
5. <virG> B09ig302 (1431)

  • Bca1363, B09ig113, and B09ig281 looked good. So i miniprepped those cultures and ran a restriction digest of two of them.

Restriction digest: first two are BamHI/XhoI and the second two are BglII/XhoI. Both parts should be in lefty:
1. ladder
2. Bca1363 BamHi/Xhoi (7620/2564)
3. <VtaA11 AtD> B09ig113 BamHI/XhoI(6820/2072)
4. Bca1363 BglII/XhoI (10184)
5. <VtaA11 AtD> B09ig113 BglII/XhoI (8892)

  • once again, everything looks single cut, what is going on?

Saturday, July 18

Pcr with oligos: ca998 and ca1172R to see if the parts are in the correct assembly vector. Ran 5K55 program. Here are the expected band sizes:
1. ladder
2. <pbad.rbs...> (4992 if in CA) (2577 if in AC)
3. <dblterm> (3669 if in CA) (1254 if in AC)

  • It looks like both parts are in the correct (CA) assembly vectors.

Sunday, July 19

Did BglII/XhoI restriction mapping. Re-did the BamHI/XhoI restriction mapping. Here are the results: Media:iGEMrestrictionweekend.pptx

Monday, July 20

After analyzing the weekend's digest we concluded that there may not be anything wrong with the vectors aside from slightly dirty minipreps.
There may also be a problem with the pBdr052KC assembly vectors, or the pir-Lefty cells. But that seems unlikely, so we set up a bunch of experiments to see what is going on.

1. PATRICK'S EXERIMENT: PCR composite parts (C09ig0025, C09ig0029, C09ig0039, C09ig0039, and C09ig0040: <displayer><dblterm>) and the displayer basic parts (B09ig57, B09ig129, B09ig306, and B09ig310) to see if the assembly worked because samples of these composite parts that were sent to sequencing failed (bad reads). 
* This experiment will check to see if assembly worked, even if the restriction maps are coming out strange
  1. pBdr052KA-C09ig0025  (819)
  2. pBdr052KC-B09ig57 (684)
  3. pBdr052KA-C09ig0029 (381)
  4. pBdr052KC-B09ig129 (243)
  5. pBdr052KA-C09ig0039 (1008)
  6. pBdr052KC-B09ig306 (873)
  7. pBdr052KA-C09ig0040 (1560)
  8. pBdr052KC-B09ig310 (1425)
  9. ladder
* Everything looks good. The parts are assembling, even though the restriction maps look wierd.
2. GABY'S EXPERIMENT: Yesterday we transformed pir-Lefty cells with plasmids that we know map well in pir-Righty to see if the problem is with the cells or the plasmids. 
We also transformed pir-Lefty with old constructs that have mapped well with BamHI/BglII before. These are BamHI/XhoI digested: 1. pBdr052AK-B09ig314 (9195/2487) - correct 2. pBdr052CA-Bca1092 (6297/2564) - very low concentration in miniprep, did not show up on the gel 3. pBdr052AK-B09ig239 (6888/2487) - correct 4. pBdr052AK-B09ig171 (7470/2487) - correct 5. Ladder 6. pBjh1601CA-Bjb21 (2345/2195) - looks like the two bands are on top of each other and it is correct 7. pBjh1601CA-Bjb27 (6042/2195) - correct 8. pBjh1601KC-Bjb7 (1998/1175) - correct 9. pBjh1601CA-Bjb28 (6048/2195) - correct Gabyexp1.jpg * It is clear that the pir-Lefty cells methylated the plasmids correctly and that everything digested cleanly. Therefore the pir-Lefty cells work well and are not the source of our problem. GABY'S SECOND EXPERIMENT: We have reason to believe that the 8-strip minipreps are not clean enough and are causing the digests to map strangely (this may also be why the sequencing is failing). So Gaby re-did the above experiment but miniprepped with the 8-strip mini-kit. 1. pBdr052CA-C09ig0019 BglII/XhoI (6162/2837) 2. pBdr052CA-C09ig0020 BglII/XhoI (6162/2837) 3. pBdr052CA-C09ig0021 BglII/XhoI (6162/3533) 4. Ladder 5. pBdr052CA-C09ig0022 BglII/XhoI (6162/3059) 6. pBdr052CA-C09ig0023 BglII/XhoI (6162/3077) 7. pBdr052AK-B09ig314 BamHI/XhoI (9195/2487) - correct lower band is very faint 8. pBdr052AK-B09ig239 BamHI/XhoI (6888/2487) - correct but extra single cut band 9. pBdr052AK-B09ig171 BamHI/XhoI (7470/2487) - correct but extra single cut band Gabyexp2.jpg * Here is proof that the minipreps are causing the crazy restriction digest problems. The same parts, miniprepped differently, showed incomplete cutting and smeary gels. So now we must begin to fine-tune the 8-strip minipreps. 3. SUSAN'S EXPERIMENT: Miniprep the newest composite parts (C09ig0019-C09ig0023) and restriction digest with BamHI/XhoI and BglII/XhoI. This experiment will to see if assembly is working between pBdr052CK and pBdr052KA plasmids. These parts should be in Righty, so here are the expected band sizes: 1. pBdr052CA-C09ig0019 BglII/XhoI (6162/2837) 2. pBdr052CA-C09ig0020 BglII/XhoI (6162/2837) 3. pBdr052CA-C09ig0021 BglII/XhoI (6162/3533) 4. pBdr052CA-C09ig0022 BglII/XhoI (6162/3059) 5. pBdr052CA-C09ig0023 BglII/XhoI (6162/3077) 6. Ladder 7. pBdr052CA-C09ig0019 BglII/XhoI (8999) 8. pBdr052CA-C09ig0020 BglII/XhoI (8999) 9. pBdr052CA-C09ig0021 BglII/XhoI (9695) 10. pBdr052CA-C09ig0022 BglII/XhoI (9221) 11. pBdr052CA-C09ig0023 BglII/XhoI (9239) Susanexp.jpg * This experiment worked really well, it looks like the assembly worked! woo! 4. SHERINE'S EXPERIMENT:Digest two Lefty-methylated pBjh1601 plasmids with BamHI/XhoI. She did this with the BamHIs and XhoIs from upstairs and downstairs to make sure all of the enzymes were working. * I dont have a picture of the gel, but all of the enzymes are working. Thanks Sherine!
  • Conclusion from all of today's experiments: The minipreps are not clean enough. Something in them is affecting the enzymes' abilities to cut completely. Although this affects restriction mapping and sequencing, it is okay for assembly.

I began a new assembly set today. See "7-9-09 iGEM09 guide.exl" for more information. Look under 7-20 assembly to see what was made.

Tuesday, July 21

Picked colonies from yesterday's assembly. Nineteen things were lawns on the LB agar strips: C8, C9, C10, D3, D4, D5, D6, D7, D8, D9, D10, D11, F3, F4, F5, F6, F7, F9, F10. These were re-streaked on new AK plates in addition to being innoculated. We picked 3 replicates into 1mL of 2YT + antibiotics in 96 well blocks. A few hours later we spotted them onto AKC plates. We also did colony PCR on a few of the colonies to be sure the assembly worked well.

Wednesday, July 22

The colony PCR looked very strange in the morning. I realized that i ran 4K55 instead of a colony PCR program on the thermocycler. This messed up the colony PCR so we did it again. Here is the colony PCR gel:
1. ladder
2. pBdr052CK-C09ig0009 (3474bp): CLONE 1 good: CLONE 2 good: CLONE 3 cotransformed
3. pBdr052CK-C09ig0001 (2295bp): CLONE 1 cotransformed: CLONE2 negative: CLONE 3 good
4. pBdr052CK-C09ig0004 (3399bp): CLONE 1 good: CLONE 2 good: CLONE 3 good
5. pBdr052CK-C09ig0016 (2214bp): CLONE 1 cotransformed: CLONE 2 good: CLONE 3 good
6. pBdr052KA-C09ig0051 (1866bp): CLONE 1 good: CLONE 2 good: CLONE 3 good
7. pBdr052KA-C09ig0082 (2544bp): CLONE 1 good: CLONE 2 questionable: CLONE 3 cotransformed

  • The clones that looked co-transformed in the colony PCR were consistent with the spot checking so we miniprepped all of the composite parts that were not co-transformed. A few of the things that came out as lawns were co-tranformed in every clone (D3-D11, F5-F7, F9), so we picked new colonies from the re-streaked plates. These things were also spot checked on AKC.

Ran a restriction digest on a few of the parts that we miniprepped, here are the expected band sizes:
1. ladder 2. C09ig0002 (BamHI/XhoI) (8436/2487) - correct
3. C09ig0003 (BamHI/XhoI) (7854/2487) - correct
4. C09ig0004 (BamHI/XhoI) (10161/2487) - correct
5. C09ig0027 (BglII/XhoI) (6577/2942) - correct
6. C09ig0024 (BglII/XhoI) (6577/3533) - correct
7. C09ig0033 (BglII/XhoI) (8577/3563) - ?

  • The bands look good, we concluded that this round of composite parts was successful. Now we need to wait for the few "catch up" parts that were lawns, and co-transformed. Hopefully these things will be ready tomorrow.

Thursday, July 23

Today we miniprepped all of the parts from 7-14 Assembly that were originally put in the wrong cell line (pBdr052KA- C09ig0024-C09ig0040 that were originally in Lefty and got transformed into Righty).

  • Sent sig107-sig112 out for sequencing.

We also began assembly of final goal parts. I assembled all the cellulases (F09ig0139-F09ig0205) and Gaby did assembly of all of the <mgfp> (or what we thought was <mgfp> and <scFv> (F09ig0001-F09ig0034 and F09ig0122-F09ig0138) parts. See .exl sheet for more information. We transformed these products into EC100D pir+ cells and plated on CA (all of the final composite parts are in pBdr052CA.
There were still a few things that came back co-transformed from the 7-20 assembly (D3 and F6). We picked new colonies of these parts and spot checked them.

Friday, July 24

The sequencing results for Sig107-Sig112 came back today. Everything looks like it was assembled correctly, except the <mgfp> protein. The sequencing reads for this came back as assembled <SOD> instead.

  • We did not do a restriction map because <mgfp> and <SOD> are to similar in size to resolve on a gel.
  • Instead we sent in more sequencing to find <mgfp> (Sig113, Sig114, and Sig117-Sig118)

We picked colonies of the Cellulase project assembly set and <SOD>/<scfv> assembly set into either LB or 2YT with CA. (we ran out of 2YT while doing this, so patrick made more)

  • We also spot checked on AKC today.

We finally have non-co-transformed clones of everything from the 7-20 assembly (all the parts we were waiting for were of the <displayer><internal passenger><terminator> type). We miniprepped them and are now ready to do the Internal Passenger Assembly Set.

Saturday, July 25

Today we assembled the Internal Passengers Project set (F09ig0035-F09ig0121).
We also made new compotent cells of EC100D.
Checked sig113, sig114, sig117 and sig118. Realized that we replaced the <mgfp> part with <SOD> in BasicAssembleRL1 and we needed to start over with this part from BasicAssembleM1.
There was very little co-transformation of the cellulase and <SOD>/<scFv> parts. We made glycerol stocks of all of these parts. We also miniprepped some of the completed cellulase and <SOD>/<scfv> parts.

  • We sent some of these in for sequencing as Sig119-Sig126.

Sunday, July 26

Today Gaby picked colonies of the Internal Passenger parts that were assembled yesterday. I spot checked them on AKC later that night.
We also transformed pBdr052AK-B09ig239 (<mgfp>) into pir-Righty so that we could begin assembly of the mussel foot protein parts soon.
Looked at the EC100D compotent cells and realized that they were spec contaminated. This is okay for the assembly we did yesterday (although not ideal) and we will be making new compotent cells tomorrow.
We realized while looking at sequencing reads (Sig119-Sig126) that not all of our final composite parts were correct. Two clones had parent vector bleed through. Sig119 (F09ig0011) and Sig126 (F09ig0205), we dont know why this is because the spot checks looked clean.

  • We also realized that for some parts the autotransporters are switched around.

Monday, July 27

Tried to do colony PCR to resolve why some of the parts showed parent vector bleed through. Maybe we just picked some bad clones? Or are all of the parts with these passengers messed up?

  • Here is the colony PCR of some of the composite parts:
1. CLONE 1: F09ig0011 (C3- SOD/scFv): composite size 3783: C09ig0001 bleed through 2295
2. CLONE 2: F09ig0011 (C3- SOD/scFv): composite size 3783: C09ig0001 bleed through 2295
3. CLONE 3: F09ig0011 (C3- SOD/scFv): composite size 3783: C09ig0001 bleed through 2295
4. CLONE 1: F09ig0125 (A4- SOD/scFv): composite size 2304 (just checking because sequencing read was short
5. CLONE 2: F09ig0125 (A4- SOD/scFv): composite size 2304
6. CLONE 3: F09ig0125 (A4- SOD/scFv): composite size 2304
7. CLONE 1: F09ig0180 (D4- Cellulase): composite size 4659 (wrong autotransporter)
8. CLONE 2: F09ig0180 (D4- Cellulase): composite size 4659
9. CLONE 1: F09ig0205 (F3- Cellulase): composite size 4482: C09ig0009 bleed through 3474
10. CLONE 2: F09ig0205 (F3- Cellulase): composite size 4482: C09ig0009 bleed through 3474
11. CLONE 3: F09ig0205 (F3- Cellulase): composite size 4482: C09ig0009 bleed through 3474
The gel looks very strange, it looks like the colony PCR didnt work. Will try it again tomorrow.

Gaby did assembly of pBdr02CK-C09ig0003 <Bca1363><mgfp>. She assembled pBdr052CA-Bca1363 in Lefty with pBdr052AK-B09ig239 in Righty. I plated on a CK plate.
The internal passenger plates spot check also looked great. Gaby tried to do colony PCR on some of the clones before we made glycerol stocks to be sure we had the correct things.

  • Here is her colony PCR:
1. CLONE 1: F09ig0035 (A1): size 2805
2. CLONE 1: F09ig0053 (B7): size 2196
3. CLONE 1: F09ig0067 (C9): size 2670
4. CLONE 1: F09ig0081 (D11): size 1983
5. CLONE 1: F09ig0099 (F4): size 3084
6. CLONE 1: F09ig0113 (G5): size 2457
Repeat with CLONE 2 and CLONE 3
This colony PCR also looks strange. Need to figure out what is going on.

Sent in iG001-iG005 for sequencing. These are final parts from the Cellulase and SOD/scFv assembly that came out weird in sequencing the first time.
We also Eco/Bam transfered Bca9494 into pBca9523 so that we can gateway this part into a pBdr052 plasmid. Bca9494 is the positive control for the strep assay and we want to be sure that it is in the same plasmid as our final parts so that it serves as a better positive control. Lane finished the Eco/Bam transfer for us last night.

Tuesday, July 28

Looked at the sequencing reads for iG001-iG005, and realized that the cel9A parts in row D of the cellulase assembly parts have switched autotransporters. The passengers look correct, but the autotransporters are all off by one. The cel6A parts that follow look correct, so the displayer mess up is probably contained in row D. This is not a huge deal because the passengers are correct. We have other things to worry about now, and will try to sort this out later.
The Eco/Bam transfer of Bca9494 into pBca9523 yielded A LOT of colonies. We will spot check them on Amp today, pick colonies, colony PCR, maybe restriction map and send for sequencing tomorrow so that we're sure that we have Bca9494 in the correct plasmid.
Need to figure out what's going on with the final parts, so i made a chart of recent sequencing results:

Part Description Clone Lefty Righty Result Analysis
F09ig0011 <Bca1363><needle scfv><espP(beta)><Bca1092> 1 C09ig0001 C09ig0034 parent: C09ig0001 ?
F09ig0024 <Bca1363><gliaden scfv><Hia AtD><Bca1092> 1 C09ig0002 C09ig0030 perfect -
F09ig0141 <Bca1363><cel3A!><c102365 AtD><Bca1092> 1 C09ig0004 C09ig0026 perfect -
F09ig0159 <Bca1363><cel5B!><vtaA11 AtD><Bca1092> 1 C09ig0005 C09ig0027 perfect -
F09ig0177 <Bca1363><cel9A!><HagAtD><Bca1092> 1 C09ig0006 C09ig0028 perfect -
F09ig0180 <Bca1363><cel9A><upaG_short><Bca1092> 1 C09ig0006 C09ig0031 wrong displayer, got <Hia AtD> (C09ig0030) instead everything in row D after D1 got shifted down one and D2 is empty
F09ig0205 <Bca1363><cel6A><yuaQ AtD><Bca1092> 1 C09ig0009 C09ig0039 parent: C09ig0009 -
F09ig0179 <Bca1363><cel9A><Hia AtD><Bca1092> 1 C09ig0006 C09ig0030 wrong displayer, got <pcryo 1125 AtD> instead confirm assumption about messed up row D
F09ig0197 <Bca1363><cel6A><upaG_short><Bca1092> 1 C09ig0009 C09ig0031 perfect probably not a flaw in cel6A
F09ig0125 <Bca1363><SOD><VtaA11 AtD><Bca1092> 1 C09ig0007 C09ig0027 perfect -

Wednesday, July 29

Looked at sequencing reads iG010-iG015. The sequencing reads for the cellulase and SOD/scfv parts are weird and need to be thought about more later. The sequencing reads for the internal passenger parts all look good, except for the things sequenced with ca56 (there are two ca56 annealing spots in these plasmids, so these oligos should no longer be used for sequencing of these parts until they are switched to another vector).

Part Description Clone Result Analysis
pBdr052CA-F09ig0205 {Pbad.rbs.prepro.StrepTag}{<cel6A!}{<yuaQ AtD>}(dblterm) 2 perfect partial, but cant see any very much of cel6A and the junction looks like a restriction site in the read, although it is sketchy by then yuAQ is okay? first clone was bad?
pBdr052CA-F09ig0028 {Pbad.rbs.prepro.StrepTag}{<gliadin binding scFv>}{<espP(beta)>}(dblterm) 1 Parent vector bleed through again, is there a problem with espP(beta) yuAQ is okay?
  • Ran Colony PCR of the internal passenger parts with ca998/G00101, here are the expected band sizes:
1. F09ig0035: CLONE 1: 2805
2. F09ig0053: CLONE 1: 2196
3. F09ig0067: CLONE 1: 2670
4. F09ig0081: CLONE 1: 1983
5. F09ig0099: CLONE 1: 3084
6. F09ig0113: CLONE 1: 2457
Repeated for CLONES 2 & 3
The colony PCR looks very strange, its odd that the colony PCR does not match the sequencing results. However i believe that these parts are correct.

Picked colonies of pBdr052CK-C09ig0003 <Bca1363><mgfp> and spot checked them on AKC so that we can miniprep them tomorrow.
Realized that the assembly vectors have a phagemid device under a Pbad promoter, which means inducing our parts would also lyse a large number of cells. This is bad. We need to move the final parts out of pBdr052 vectors. To do this we are EcoRI/BamHI transferring lots of the intermediates into pBca9495 assembly vectors.

  • We are moving the parts as noted on "pBca9495 Beginnings" in the iGEM09 Parts google doc.
  • We moved all of the simple intermediates today into pBca9495AK (lefty) and pBca9495KC (righty) we still need to figure out a scheme to move the more complicated internal passenger intermediates
  • I want to do pBca9495KA (lefty for <displayer>) and pBca9495AC (righty for <internal pass><terminus>) but i need to find pBca9495AC plasmids in order to do this.

Today i transformed pBca9495AC-M10301, pBca9495AC-M10301, and pBca9495KA-M10322 into DH10B. These plasmids were previously in righty, and i wanted to remove their methylation so that i can EcoRI/BamHI digest them.
I also made glycerol stocks of the finished internal passenger parts in pBdr052.

Thursday, July 30

Miniprepped pBca9495AC-M10301, pBca9495AC-M10301, pBca9495KA-M10322, and pBdr052CK-C09ig0003 <Bca1363><mgfp>. Every clone of pBdr052CK-C09ig0003 <Bca1363><mgfp> was good (nothing co-transformed). Did an analytical digest on pBdr052CK-C09ig0003, and vector backbone digests on pBca9495AC-M10301, pBca9495AC-M10301, and pBca9495KA-M10322.
The EcoRI/BamHI transfers of all the Lefty (pBca9495AK) parts look great (lots of nice big colonies), but the EcoRI/BamHI transfers of the Righty (pBca9495KC) parts are pretty small. I am going to wait until the afternoon to pick colonies so that we do not pick red colonies by accident. We will pick two clones of each into 1mL 2YT, and miniprep everything tomorrow. We will also need to restriction map to make sure the parts are the correct sizes.

Friday, July 31

Realized that the EcoRI/BamHI transfers of the Righty (pBca9495KC) were probably wrong because the things going into those digests were BamHI methylated. Need to transform those parts into a non-methylating pir strain in order to remove their methylation. Then re-do the EcoRI/BamHI transfers.

  • Transformed C09ig00027, C09ig0035, C09ig0024, C09ig0025, C09ig0026, C09ig0028, C09ig0029, C09ig0030, C09ig0031, C09ig0032, C09ig0033, C09ig0034, C09ig0036, C09ig0037, C09ig0039, C09ig0040 into pir116+ for E/B transferring tomorrow. And transformed C09ig0019, C09ig0020, C09ig0021, C09ig0022, and C09ig0023 into pir116+ for E/B transfer at a later date.

Looked at sequencing of iG016 and realized that we picked a messed up clone of pBdr052CK-C09ig0003 and needed to miniprep new clones. So i miniprepped two new clones of C09ig0003 and restriction digested them. Here is that digest (EcoRI/BamHI):

1. pBdr052CK-C09ig0003 Lefty: 8640/1701
2. pBdr052CK-C09ig0003 Lefty: 8640/1701
* The restriction map of these things looks great. We should be able to use these mgfp parts, but we will sequence CLONE 2 as iG017.

Miniprep EB transfers: Eb731.jpg

August 2009

Monday, August 3

Miniprepped the pBca9495KA-Bca1144#5 (DH10B), pBca9495AC-Bca1144#5 (DH10B), pBca9495AK-Bca1363 (LEFTY), and pBca9495AK-Bca9494 (DH10B) but not pBca9495AK-C09ig0003 (LEFTY) because we found out that the assemblies were unsuccessful. Then i ran a restriction map to make sure these parts were correct.

  • Here is the digest (EcoRI/BamHI):
1. pBca9495KA-Bca1144#5 (3171/910): good
2. pBca9495AC-Bca1144#5 (3039/910): good
3. pBca9495AK-Bca1363 (3171/1467): good 
4. pBca9495AK-Bca9494 (3171/2188): to high? 
  • Because the maps of the new vectors (pBca9495KA-Bca1144#5 and pBca9495AC-Bca1144#5) looked good, Susan and Sherine began to EcoRI/BamHI transfer some of our parts (C09ig0019-C09ig0023, and the B09ig parts noted in "pBca9495 Beginnings") into these vectors.

BseRI/XhoI digest of EcoRI/BamHI transferred parts:

1. pBca9495KC-C09ig0027 (3361): Questionable  
2. pBca9495KC-C09ig0035 (4348)
3. pBca9495KC-C09ig0024 (3952)
4. pBca9495KC-C09ig0025 (3802) 
5. pBca9495KC-C09ig0026 (3946) 
6. pBca9495KC-C09ig0028 (3361)
7. pBca9495KC-C09ig0029 (3364) 
8. pBca9495KC-C09ig0030 (3361) 
9. pBca9495KC-C09ig0031 (3388) 
10. pBca9495KC-C09ig0032 (3982) 
11. pBca9495KC-C09ig0033 (3982) 
12. pBca9495KC-C09ig0034 (4471) 
13. pBca9495KC-C09ig0036 (3595) 
14. pBca9495KC-C09ig0037 (4693): Wrong, looks like mutated Bca1144#5 
15. pBca9495KC-C09ig0039 (3991) 
16. pBca9495KC-C09ig0040 (4543) 
If it were pBca9495KC-Bca1144#5 mutant: 2328/1212/34

EcoRI/XhoI digest of EcoRI/BamHI transferred parts:

1. pBca9495KC-C09ig0027 (2228/1133): Wrong looks like Bca1144#5 
2. pBca9495KC-C09ig0035 (3215/1133) 
3. pBca9495KC-C09ig0024 (2819/1133) 
4. pBca9495KC-C09ig0025 (2669/1133) 
5. pBca9495KC-C09ig0026 (2813/1133) 
6. pBca9495KC-C09ig0028 (2228/1133) 
7. pBca9495KC-C09ig0029 (2231/1133) 
8. pBca9495KC-C09ig0030 (2228/1133) Questionable, maybe to big?
9. pBca9495KC-C09ig0031 (2255/1133) 
10. pBca9495KC-C09ig0032 (2849/1133) 
11. pBca9495KC-C09ig0033 (2849/1133) 
12. pBca9495KC-C09ig0034 (3338/1133) 
13. pBca9495KC-C09ig0036 (2462/1133)
14. pBca9495KC-C09ig0037 (3560/1133): Wrong, looks like mutated Bca1144#5
15. pBca9495KC-C09ig0039 (2858/1133) 
16. pBca9495KC-C09ig0040 (3410/1133)
If it were pBca9495KC-Bca1144#5: (2751/1133) 
  • Gaby miniprepped new clones of C09ig0027, C09ig0030, and C09ig0039 and set up new restriction digests of these parts. We will run the gel for them tomorrow and hopefully begin assembly with these parts then too.

I gave Sherine and Susan the task of figuring out what is happening to C09ig0003 (<Bca1363><mgfp>). I think the problem may have been transforming it into pir-RIGHTY and then going strait into liquid culture instead of plating, picking clones, and looking for co-transformation.

  • I gave them the pBdr052AK-B09ig239 from BasicAssembleM1 which should be un-methylated, to re-transform into pir-RIGHTY. I also gave them a miniprep Gaby and i did previously (7-27-09) which should be a methylated version of the strain to re-transform in parallel.
  • Hopefully this time the assembly works.

Tuesday, August 4

Ran restriction digests of new clones of:

1. pBca9495KC-C09ig0027: BseRI/XhoI: (3361): good
2. pBca9495KC-C09ig0030: BseRI/XhoI: (3361): no
3. pBca9495KC-C09ig0037: BseRI/XhoI: (4693): good
4. Ladder
5. pBca9495KC-C09ig0027: EcoRI/XhoI: (2228/1133): good
6. pBca9495KC-C09ig0030: EcoRI/XhoI: (2228/1133): no
7. pBca9495KC-C09ig0037: EcoRI/XhoI: (3560/1133): good
  • Sherine is miniprepping the 3rd clone of pBca9495KC-C09ig0030, hopefully this one will be correct. If not we will need to pick new colonies.

Did assembly of the pBca9495AC Cellulase and scFv/leucine zipper parts. See "9495 Assembly" in "7-9-09 iGEM09 guide1" for more info.
Picked colonies of the last round of EcoRI/BamHI transfers: C09ig0019-C09ig0023 and B09ig1-B09ig310.

Wednesday, August 5

Learned how to use the dishwasher for 96/24 well blocks. Run "medium" cycle and autoclave on fast cycle (sterilize 25 min, exhaust 15).
Looked at the strips from yesterday's assembly. We have colonies for everything, but a bunch of strips have very few colonies:

#F09ig0154 {Pbad.rbs.prepro.StrepTag}{<cel3A!}{<TshA>}(dblterm)
#F09ig0164 {Pbad.rbs.prepro.StrepTag}{<cel5B!}{<CPG_L6>}(dblterm)
#F09ig0171 {Pbad.rbs.prepro.StrepTag}{<cel5B!}{<TshA>}(dblterm)
#F09ig0181 {Pbad.rbs.prepro.StrepTag}{<cel9A!}{<CPG_L6>}(dblterm)
#F09ig0198 {Pbad.rbs.prepro.StrepTag}{<cel6A!}{<CPG_L6>}(dblterm)

scFv/Leucine zippers:

#F09ig0009 {Pbad.rbs.prepro.StrepTag}{<type IIIs Needle Complex scFV>}{<CPG_L2>}(dblterm)
#F09ig0016 {Pbad.rbs.prepro.StrepTag}{<type IIIs Needle Complex scFV>}{<yuaQ AtD>}(dblterm)
#F09ig0033 {Pbad.rbs.prepro.StrepTag}{<gliadin binding scFv>}{<yuaQ AtD>}(dblterm)
#F09ig0043 {Pbad.rbs.prepro.StrepTag}{<KILR>}{<CPG_L2>}(dblterm)
#F09ig0050 {Pbad.rbs.prepro.StrepTag}{<KILR>}{<yuaQ AtD>}(dblterm)
#F09ig0060 {Pbad.rbs.prepro.StrepTag}{<EILD>}{<CPG_L2>}(dblterm)
#F09ig0076 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<upaG_short>}(dblterm)
#F09ig0084 {Pbad.rbs.prepro.StrepTag}{<GS5-IILK>}{<yuaQ AtD>}(dblterm)

We can see a pattern with <CPG_L2> and <yuaQ AtD> so we will be careful with those parts.

  • We picked colonies of the Cellulase and scFv/Leucine zippers.

Taught Susan and Sherine how to miniprep with the 8-column strips. We miniprepped the last set of EcoRI/BamHI transfers.

Thursday, August 6

Restriction digest of last EcoRI/BamHI transfers into pBca9495:
EcoRI/XhoI digest:

  1. pBca9495AC C09ig0019 (2320/1001) weird and very high bands
  2. pBca9495AC C09ig0020 (2329/1001) very faint, may have correct band sizes and a monocut
  3. pBca9495AC C09ig0021 (3016/1001) looks okay
  4. pBca9495AC C09ig0022 (2542/1001) part band a little high? maybe okay
  5. pBca9495AC C09ig0023 (2560/1001) too many bands, both part bands look a little high
  6. pBca9495KA B09ig1 (2818/1196) part band is way to big
  7. pBca9495KA B09ig57 (2668/1196) looks good
  8. pBca9495KA B09ig79 (2812/1196) looks good
  9. ladder
  10. pBca9495KA B09ig121 (2227/1196) looks good
  11. pBca9495KA B09ig129 (2230/1196) looks good
  12. pBca9495KA B09ig137 (2227/1196) looks good
  13. pBca9495KA B09ig277 (2254/1196) looks good
  14. pBca9495KA B09ig278 (2848/1196) small
  15. pBca9495KA B09ig279 (2848/1196) looks good
  16. pBca9495KA B09ig113 (2227/1196) looks good
  17. pBca9495KA B09ig280 (3337/1196) looks good
  18. ladder
  19. pBca9495KA B09ig281 (3214/1196) looks good
  20. pBca9495KA B09ig282 (2461/1196) no
  21. pBca9495KA B09ig283 (3559/1196) looks good
  22. pBca9495KA B09ig306 (2857/1196) small, but okay? frowning?
  23. pBca9495KA B09ig310 (3409/1196) small, but okay? frowning?

If digested pBca9495AC-Bca1144#5 (2948/1001) BseRI/XhoI digest:
Redo with EcoRI/BamHI:

  1. pBca9495KA B09ig1 (3171/843)
  2. pBca9495KA B09ig57 (3171/693)
  3. pBca9495KA B09ig79 (3171/837)
  4. pBca9495KA B09ig121 (3171/252)
  5. ladder
  6. pBca9495KA B09ig129 (3171/255)
  7. pBca9495KA B09ig137 (3171/252)
  8. pBca9495KA B09ig277 (3171/279)
  9. pBca9495KA B09ig278 (3171/873)
  10. ladder
  11. pBca9495KA B09ig279 (3171/873)
  12. pBca9495KA B09ig113 (3171/252)
  13. pBca9495KA B09ig280 (3171/1362)
  14. pBca9495KA B09ig281 (3171/1239)


Friday, August 7

Saturday, August 8

Did Internal Passenger Intermediates/mgfp assembly (see 7-9-09 iGEM09 guide: 9495 Assembly).
Also made more cam antibiotic: 34mg/mL in ethanol.

Monday, August 10

Picked colonies of 8-8-09 Internal Passenger Intermediates/Mgfp Assembly. Picked 3 clones into 1mL of 2YT and AC. In CLONE 3:D9 and D10 are D9, in CLONE 3: something in Rows A and B may be co-contaminated (toothpick fell), in CLONE 3: G5 and G6 are switched, H6 (H6 should not have anything growing in it) and E1 had very few colonies growing.

Tuesday, August 11

Realized, after beginning to miniprep, that the internal passenger intermediates were put into DH10B (not RIGHTY) and plated on AC (not KC) and therefore were not the correct products. We re-did that assembly today, along with the passenger-less displayer final parts. See .exl sheet for more info.
Miniprepped a few clones of the mgfp final parts and restriction mapped them (see gaby's notebook). The restriction maps look good so we sent the parts out for sequencing.

  • I also made frozen cell stocks of the mgfp parts (two clones) and innoculated them for the strep assay that Susan and Sherine will do tomorrow.

Wednesday, August 26

Over the weekend, susan and sherine finished Eco/Bam transferring all of the parts needed to make the linker set. The linkers (M10022, M10089, M10025, M10087, and 14-mer Gly-Ser repeats) were transferred from pBca9523 to pBca9495CK. The Bca1363.mgfp part (C09ig0003) was transferred from pBca9495AK to pBca9495AC. Everything was spot checked. The spots looked good, so i miniprepped the first set of clones today and restriction digested them with EcoRI/XhoI. (will run the gel tomorrow)

Thursday, August 27

The restriction map of the e/b transferred linker intermediate parts looks good. EcoRI/XhoI digest:

1.pBca9495AC-C09ig0003: 1196/3542
2.pBca9495CK-M10087: 1001/2287
3.pBca9495CK-M10022: 1001/2395
4.pBca9495CK-M10089: 1001/2662
5.pBca9495CK-14mer: ?
6.pBca9495CK-M10025: 1001/2740

Started assembly of these parts because the restriction map looked good.

Sunday, August 29

Miniprepped the clones of C09ig0114-C09ig0118 that were not co-transformed and restriction mapped them with EcoRI/XhoI:
Helped a bit with the AIDA/mgfp assays. AIDA alone auto-aggregated once induced and put into DH10B. We took some OD600 measurements to quantify how much it auto aggregated:

  1. AIDA-No pass: 0.942
  2. AIDA-mgfp5: 1.816


Tuesday, September 8

Waiting on info from sherine to start the assembly of cellulases/scfv with linkers. Also need to start the cellulases with no displayer and the internal passenger parts.
Ran the internal pass intermediates after Eco/Xho digest and decided that we need to re-E/B transfer those parts, they came out to large:

#pBca9495AC-C09ig0019 (2123/1196)
#pBca9495AC-C09ig0020 (2132/1196)
#pBca9495AC-C09ig0021 (2819/1196)
#pBca9495AC-C09ig0022 (2345/1196)
#pBca9495AC-C09ig0023 (2363/1196)

Thursday, September 10

Plated assembly of the cellulase-no-displayer parts (F09ig0350-F09ig0353) and the cellulase/scfv linker intermediates. Also plated the EcoRI/BamHI transfer of C09ig0019-C09ig0023 from pBdr052 to pBca9495AC.

Sunday, September 13

Lots of problems today. :(

  • Made frozen stocks of the cellulase negative controls.
  • Miniprepped the cellulase/scfv linker intermediates using the 8-strip minis. Then i did a restriction digest (EcoRI/Bam) to see if the products were the correct size. The digest looked terrible (everything was only one band). I was going to continue with assembly anyway (because i'd already made the reaction plate) but one of the tubes had to little DNA. I decided to re-transform that one instead and will do the assembly tomorrow after i can send things out for sequencing.
  • Also finished the Eco/bam transfers of the internal pass intermediates. Plated them on AC and will hopefully have things that grow tomorrow. Used unmehtylated things for that this time.

Saturday, September 19

Finished assembly of Internal Passenger intermediates yesterday, so i came in to pick colonies today. This time the assembly set should have worked perfectly because all of the parent parts were checked before they were used in the assembly. However, a few of the strips had very few colonies growing. Here are the assembly stats:

  1. 64 products
  2. plated ~150uL of cell reaction mixture (except for row B: ~100uL)
  3. rescue 45 min
  4. 50uL comp cells/rxn
  5. A1-A12 almost a lawn
  6. B1-B12 (except B1) lots of colonies
  7. C1-F5 very few colonies (E7-F5 ~3 colonies on many strips)
  8. D6= 2 colonies
  9. F2= 1 colony!!
  10. clone 2: D4&5 switch

Also, have been trying to make sure the mgfp-linker parts are okay. Minipreps looked wierd:
MGFP-linkers= row1-5 (eco/bam digest):

1. pBca9495AC-F09ig0284
2. pBca9495AC-F09ig0306
3. pBca9495AC-F09ig0314
4. pBca9596AC-F09ig0337
5. pBca9495AC-F09ig0351

Internal Pass Intermediate Parents:

7. pBca9495AC-C09ig0023(eco/xho) (2123/1196)
8. pBca9495AC-C09ig0022-2 (eco/xho) (2132/1196)
9. pBca9495AC-C09ig0022-1 (eco/xho) (2819/1196)
10. pBca9495AC-C09ig0022-2 (bseri/xho) (2345/1196)
11. pBca9495AC-C09ig0022-1 (bseri/xho) (2363/1196)

The one high band is bad, i tried sending the mgfp-linker final parts out for sequencing, but all of the sequencing came back bad. Today im going to grow up more of these bacteria and try the miniprepping again tomorrow.

Tuesday, September 22

Internal Pass intermediates assembly set info:
C2-F5 are co-transformed. These are all of the caspace, cub, and nub parts. I took another look at the sequencing data, and the backbones for these parts do not look correct. I am going to throw them out of the set and focus on the tev parts. Of the 26 tev parts:

  1. 4 were co-transformed in clone 1 (including 2 bad AIDA parts)
  2. 9 were co-transformed in clone 2 (including 2 bad AIDA parts)

Here is an Eco/Xhoi digest of some of the parts:

1. ladder
2. pBca9495KC-C09ig0041 (3197/1133)
3. pBca9495KC-C09ig0056 (3236/1133)
4. pBca9495KC-C09ig0065 (2615/1133)
All look good. Continue with assembly! :)

Now im going to miniprep the non-co-transformed clones and assemble them with Bca1363.INP

Wednesday, September 23

Picked colonies of the tev internal pass assembly. There were 4 strips where nothing grew.
Tomorrow need to spot check, send e/b transfered cellulase linker intermediates for sequencing (rm first) >> if those look good i can start a new assembly for those on friday.

Friday, September 25

Checked the E/B transfer products (that went into the failed assembly) pBca9495AC-C09ig0001, C09ig0004, C09ig0006. Here is the gel (EcoRI/BamHI digest):

#pBca9495AC-Bca1144#5 (to check backbone size against)
#pBca9495AC-C09ig0001: 3037/2304
#pBca9495AC-C09ig0004: 3037/4008
#pBca9495AC-C09ig0006: 3037/4263

250px everything looks good except needle scfv. I'm going to send them for sequencing because the assembly products were all bad and i'm not sure why. prehaps the backbone is messed up?

Also did spot check of tev internal pass intermediates.

Monday, September 29

Looked at sequences of iG096-iG098. The two cellulase parts look good, the scfv does not. Need to re-E/B transfer the scfv. Will start assembly again with the cellulase parts.

October 2009

Monday, October 5

Looked at PCR of pBca9495AK-C09ig00120, C09ig0129 (w/G00101 and ca998) today and it didnt look good. The PCR did not work, there were no bands. I am going to re-miniprep the parent parts (by re-transforming them into righty and lefty) and maybe do the PCR again.

Wednesday, October 7

Yesterday i retransformed pBca9495AC-C09ig0001, 4, and 6 into lefty. I also retransformed pBca9495CK-M10022, 89, 25, 87 and L10001 into lefty. I miniprepped them to get new clones because we were suspicious that the minipreps were bad somehow. I restriction mapped them all with EcoRI/XhoI and here are the results:

#pBca9495AC-C09ig0001 (4145/1196)
#pBca9495AC-C09ig0004 (5849/1196)
#pBca9495AC-C09ig0006 (6104/1196)
#pBca9495CK-M10022 (2395/1001)
#pBca9495CK-M10089 (2662/1001)
#pBca9495CK-M10025 (2740/1001)
#pBca9495CK-M10087 (2287/1001)
#pBca9495CK-L10001 (2032/1001)
  • If these were Bca1144#5 mutants they would be at (2885/1001)

Gel100709.jpg It looks like the linker parts may have been co-transformed. I am going to re-do the E/B transfer today.

Friday, October 9

Came in to pick colonies of the pBca9495CK-M10022 to L10001 E/B transfers. There is a little colony-big colony problem on the plate. I am going to pick the big colonies.

Sunday, October 11

I restriction mapped pBca9495CK-M10022 to L10001 with EcoRI/XhoI and here are the expected band sizes:

#pBca9495CK-M10022 (2395/1001)
#pBca9495CK-M10089 (2662/1001)
#pBca9495CK-M10025 (2740/1001)
#pBca9495CK-M10087 (2287/1001)
#pBca9495CK-L10001 (2032/1001)