Week 3

Wed (5/29/2019) Took inventory of lab
Ripal, Jemy, Victor
Wed (5/29/2019) Autoclaved pipette tips & eppendorf tubes
Jemy, Victor
Fri (5/31/2019) Autoclaved LB & LB agar
Victor, Jemy, Ripal
Fri (5/31/2019) Transformed E. coli with plasmids pBAD02 and pskDuet1
Plasmids obtained from graduate student Dylan Tomares in the Childer's lab.
Sat (6/1/2019) Miniprepped plasmids
Sat (6/1/2019) Colony PCR
Sat (6/1/2019) Started liquid cultures
(PBAD02 Ampicillin, pskDuet1 Kanamycin, other pTEV Ampicillin)
Sat (6/1/2019) Created freezer stock for pBAD02, pskDuet1

Week 4

Sun (6/2/2019) Linearized pTEV backbone
Linearized gel

Week 6

Wed (6/19/2019) Received set 1 gBlocks from IDT
Wed (6/19/2019) Cloned set 1 gBlocks into pTEV5 and transformed DH5α cells
DH5α colonies following Gibson assembly
Thu (6/20/2019) Colony PCR of set 1 constructs

Colony PCR - 1

Top: pVS28:(3,4,5,6,7,8,1), pVS29:(1,2,3,4), pVS43:(1,2,3,4), pVS29:(5)

Bottom: pVS29:(6), pVS30:(1,2,3,4,5,6), pVS31:(1,2,3,4,5,6,7,8)

Colony PCR - 2

Top: pVS34:(1,2,3,4,5,6,7,8), pVS33:(1)

Bottom: pVS33:(2,3,4,5,6,7,8), pVS28:(1,2)

Fri (6/21/2019) Made DH5α & BL21 comp cells
Victor, Harrison
Fri (6/21/2019) Miniprepped set 1 constructs to prepare for BL21 transformation
Victor, Harrison
Fri (6/21/2019) Transformed set 1 constructs into BL21 for expression
Victor, Harrison

BL21 transformation plates

(slightly overgrown)

Week 7

Sun (6/23/2019) Started liquid cultures of set 1 constructs in BL21
Mon (6/24/2019) Small scale expression test

Expression gel (SDS-PAGE)

Left to right: pVS(28,29,30,31,33,34,43)

Our ladder was old and did not work

(we got better at running gels I promise)

Mon (6/24/2019) Prepared buffers for protein purification
Victor, Harrison
Tue (6/25/2019) Test purification of pVS33 with Ni-NTA affinity chromatography
Dylan Tomares, a graduate student from the Childer's lab assisted us with purification of pVS33.

Purification gel (SDS-PAGE)

Insufficient lysis caused protein to be retained in the pellet

Tue (6/25/2019) Started overnight liquid cultures for miniprep
Wed (6/26/2019) Miniprepped cultures for sequencing
Ripal, Jemy, Mel, Harrison
Plasmid concentration was insufficient for Sanger sequencing.
Wed (6/26/2019) Started overnight liquid cultures for miniprep
Thu (6/27/2019) Miniprepped cultures and submitted for sequencing
Thu (6/27/2019) Expressed, induced and pelleted two 250mL cultures of pVS33
Victor, Harrison
Fri (6/28/2019) Attempted purification of pVS33
Victor, Harrison
Unable to lyse cells completely with sonic dismembrator.

Week 8

Mon (7/1/2019) Sonic dismembrator tests

We tried several different cycle patterns on the sonic dismembrator to determine the best mode of lysis moving forward.

Dismembrator gel (SDS-PAGE)

L=ladder, P=pellet, S=supernatant

  • 1a - (10 sec on/20 sec off) x 20
  • 1b - (10 sec on/20 sec off) x 40
  • 2a - (15 sec on/15 sec off) x 20
  • 2b - (15 sec on/15 sec off) x 40
  • 3a - (25 sec on/5 sec off) x 15
  • 4 - (1.5 min on)
  • C - no lysis
Tue (7/2/2019) Expressed TEV protease and pVS43

Week 9

Mon (7/8/2019) Small scale expression test

We attempted another small scale expression test. Cells were grown to OD600=0.5 and induced to 1mM IPTG for 4 hours at room temperature.

Expression test gel (SDS-PAGE)

For each construct, left=uninduced, right=induced

Mon (7/8/2019) TEVp purification attempt

Starting with a 250mL culture of TEVp, we attempted Ni-NTA purification again using our new lysis protocol. Unfortunately, almost all of the protein was lost in the flow-through indicating poor his-tag binding or a faulty column/protocol.

Purification test gel (SDS-PAGE)

M=marker, P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

Tue (7/9/2019) Transformation of set 2

We received cloned-in plasmids from Twist with out set 2 constructs and transformed them into BL21.

Wed (7/10/2019) Purification tests
Harrison, Jemy, Ripal

We took 20mL cultures of pVS29, pVS31 and pVS33 and attempted Ni-NTA purification. However, based on an A280 reading, we observed no protein in our elution -- likely due to excessive washing.

Fri (7/12/2019) Expression of pVS(29,31,33)
Harrison, Jemy, Ripal

We expressed 250mL cultures of constructs pVS(29,31,33) for further purification tests.

The lower band for each construct is our protein of interest and the top band is LacI.

Expression gel (SDS-PAGE)

For each, left=uninduced, right=induced

Week 10

Tue (7/16/2019) Purification of pVS(31,33)
Harrison, Jemy, Ripal

We tried purification of pVS31 and pVS33. Unfortunately most of our protein was retained in the pellet indicating insolubility.

Purification gel (SDS-PAGE)

P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

Tue (7/16/2019) Purification of pVS(31,33) with Emulsiflex

Attempted purification of pVS31 and pVS33 with an Emulsiflex. Most of the protein was retained in the pellet indicating solubility (as above).

Purification gel (SDS-PAGE)

Week 11

Tue (7/23/2019) Small scale expression tests

Since our previous expression tests were still relatively uncertain, we tried incubating to a higher OD (0.5-0.6) and expressing for 5 hours at room temperature.

We expressed set 1 constructs pVS(28,29,30,31,33,34,43) as well as our newly transformed set 2 constructs pVS(20,22,23,24,25,26).

Everything expressed at the expected size except for pVS28 (which did not express). After looking back at the sequencing results, we realized that our Gibson assembly introduced a deletion in the reading frame leading to a frame shift mutation.

Expression gel (SDS-PAGE)

  • pVS20: 32.02 kDa
  • pVS22: 21.44 kDa
  • pVS23: 14.90 kDa
  • pVS24: 32.02 kDa
  • pVS25: 13.62 kDa
  • pVS26: 20.75 kDa

Expression gel (SDS-PAGE)

  • pVS28: 19.86 kDa
  • pVS29: 18.80 kDa
  • pVS30: 18.69 kDa
  • pVS31: 24.86 kDa
  • pVS33: 19.91 kDa
  • pVS34: 21.85 kDa
  • pVS43: 21.44 kDa
Wed (7/24/2019) Extract inserts via PCR

Since several of our constructs appeared to be relatively insoluble, we decided to try cloning our inserts into pTEV6 which expresses maltose binding protein as an N-terminal fusion tag.

To extract our insert, we ordered insertion primers and ran PCR followed by gel electrophoresis. Then we cut out the corresponding bands and extracted the DNA.

Insertion bands

Top: pVS(28,29,30,31)

Bottom: pVS(33,34,43)

Fri (7/26/2019) Expression of pVS(29,30,34,43)
Victor, Ripal, Jemy, Mel

We expressed 250mL cultures of pVS(29,30,34,43) and pelleted to prepare for further purification.

Sat (7/27/2019) Purification of pVS(30,34,43)
Victor, Ripal, Jemy, Mel

We attempted Ni-NTA purification of three constructs and finally figured out the protocol.

All three constructs expressed and showed up in the elution with minimal contaminants.

Ni-NTA purification of pVS30 and pVS34

Ni-NTA purification of pVS43

Week 12

Mon (7/29/2019) Purification of pVS29
Victor, Ripal, Jemy, Mel

Ni-NTA purification of pVS29

Tue (7/30/2019) Gibson assemblies + transformation of DH5α
We received gene fragments for set 3 from Twist and cloned them into pTEV5. Also, we took the extracted inserts of set 1 and cloned them into pTEV6 to express an N-terminal MBP fusion protein.
Wed (7/31/2019) Colony PCR verification
Inserts were verified via colony PCR.
Thu (8/1/2019) Purification of pVS22 and pVS23
Ripal, Jemy

Ni-NTA purification of pVS22(left) and pVS23(right)

pVS22 seems to be relatively insoluble.

Week 13

Mon-Fri (8/5/2019)-(8/9/2019) Expression and purification of set 2 + pVS29
Harrison, Ripal

We were able to purify all the cosntructs with varying levels of purity.

U=uninduced, I=induced, P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

Ni-NTA purification of pVS20 and pVS26 (SDS-PAGE)

Ni-NTA purification of pVS22 and pVS23 (SDS-PAGE)

Ni-NTA purification of pVS24 and pVS25 (SDS-PAGE)

Ni-NTA purification of pVS29 (SDS-PAGE)

Tue (8/8/2019) Retry colony PCR of pTEV6 constructs + set 3
Harrison, Ripal

After observing no colonies growing on AMP+ LB plates, we decided to retry colony PCR to verify the insertion.

Unfortunately, we didn't see a single positive hit from these colonies, indicating that cloning did not work.

Colony PCR of pVS36 and pVS37 (+pVS21)

Colony PCR of pVS38 and pVS39 (+pVS21)

Colony PCR of pVS40 and pVS41

Colony PCR of pVS48 and pVS50

Thu (8/10/2019) Lysate tests
Harrison, Ripal

We attempted to mix whole-cell lysates containing each of our intein halves to try to observe intein splicing.

However, background cellular proteins made it very difficult to identify any bands that were formed or depleted.

In the future, we could try using a His-tag stain to identify our synthetic protein constructs (and reactants).

Fri (8/11/2019) Sequencing of pVS28

Miniprepped two other colonies of pVS28 and sent in for sequencing.

Week 14, 15, 16

Everyone is on break

Week 17

Wed (9/4/2019) Test purification with BugBuster
Ripal, Jemy

Attempted purification of pVS33 with BugBuster.

Protein did not show up in the elution.

Purification of pVS33 (SDS-PAGE)

Week 18

Mon (9/9/2019) PCR cloning of set 1 + Gibson assemblies

Attempted Gibson cloning of our set 1 constructs in pTEV6 and verified insertions via colony PCR.

Mon (9/9/2019) Transformation of CSL constructs

Transformed several constructs from CSL into BL21 for expression.

Wed (9/11/2019) Transformed DH5α

Gibson assemblies were transformed into DH5α comp cells.

Fri (9/13/2019) Made BL21 comp cells

Prepared more BL21 comp cells for future transformations.

Sat (9/14/2019) Transformation + colony PCR of sspDnaB iGEM blocks
Ripal, Jemy

Two constructs encoding sspDnaB split-inteins were transformed into E. coli BL21 for expression and colony PCR was used to verify inserts.

Colony PCR

Sat-Sun (9/14/2019)-(9/15/2019) Test purification with BugBuster
Ripal, Jemy

Attempted purification of pVS29 and pVS30 with BugBuster and sonic dismembrator as a control.

Gel overstained, it is difficult to tell which method worked better.

Week 19

Mon (9/16/2019) Miniprepped all constructs + re-transformation of DH5α

Plates containing our existing constructs were transported to our secondary workspace in Dr. Childer's lab in order to make use of their equipment. These constructs were miniprepped and retransformed into DH5α comp cells.

Wed (9/18/2019) DH5α & BL21 transformations

DH5α constructs from 9/16 were miniprepped and transformed into BL21 for expression.

Additonally a second set of transported constructs was miniprepped and transformed into DH5α.

Thu (9/19/2019) Remaining BL21 transformations

The second set of transported constructs was minipreppe from DH5α colonies and transformed into BL21 comp cells.

Thu (9/19/2019) Cloning of split-linker set and transformation into DH5α
Victor, Harrison

We received gBlocks encoding our split-linker constructs from IDT and cloned them into a pTEV6 backbone. Gibson assemblies were transformed into E. coli DH5α cells and plated on LB media. We also plated several previous assemblies of our pTEV6 variants of set 1.

Fri (9/20/2019) Freezer stock

Made freezer stock of all the transported BL21 colonies for future expression.

Fri (9/20/2019) Colony PCR

Performed colony PCR on the newly transformed constructs in order to screen for insertions.

Used pTEV6 primers provided by a graduate student in Dr. Childer's lab.

Bold/underlined colonies indicate positive hits.

Colony PCR 1

Colony PCR 2

Sat (9/21/2019) Purification of pVS34 and pVS43
Ripal, Jemy

Purification of pVS34 (left) and pVS43 (right) (SDS-PAGE)

Week 20

Mon (9/23/2019) Miniprepped positive colonies

Overnight colonies of the positive hits were miniprepped to prepare for sequencing.

Wed (9/25/2019) Ordered pTEV6 screening/sequencing primers

Designed and ordered four new pTEV6 screening and sequencing primers to get better colony PCR results and prepare for Sanger sequencing.

Fri (9/27/2019) Colony PCR test with new primers

Received primers from IDT and tested each combination of F/R primers on four constructs of pHG44.

All colonies showed strong positive hits.

Colony PCR

Sat (9/28/2019) Repeat colony PCR

Redid colony PCR of the previous constructs using the new pTEV6 primers.

Colony PCR 1

Colony PCR 2

Sun (9/29/2019) Miniprepped and transformed BL21

All the colonies with positive hits were miniprepped from overnight cultures and transformed into E. coli BL21 for expression.

BL21 plates

Week 21

Mon (9/30/2019) Expression of pVS22

Large scale expression of pVS22 for lysate mixture tests.

Tue (10/1/2019) Freezer stock of new constructs

Created freezer stock of newly transformed BL21 constructs from overnight cultures.

Tue (10/1/2019) Small scale expression tests

Grew 4mL cultures of the new constructs to OD600=0.5-0.6 and induced with 1mM IPTG for 4 hours.

All the constructs expressed protein at the expected size.

L=ladder, U=uninduced, I=induced

Expression test (SDS-PAGE)

Expression test (SDS-PAGE)

Wed (10/2/2019) Expression of pVS23

Large scale expression of pVS23 for lysate mixture tests.

Fri (10/4/2019) His stain test + lysate assay of pVS22/pVS23

We received a his-stain that can be applied to SDS-PAGE gels to visualize His-tagged proteins.

We tested the stain on a lysate mixture of pVS22 and pVS23.

Unfortunately we were unable to get access to the gel imager over the weekend and the gel destained considerably leaving faint bands.

Lysate mixture of pVS22 and pVS23 (Coomassie brilliant blue) (SDS-PAGE)

Lysate mixture of pVS22 and pVS23 (InVision His-tag stain) (SDS-PAGE)

Sun (10/6/2019) Colony PCR of sspDnaB
Mel, Jemy

Ran colony PCR screening on several sspDnaB constructs from the 2019 iGEM distribution kit.

Week 22

Mon (10/7/2019) Expression test of pHG68

Tried expressing pHG68 at room temperature and 37 C to see if it would automatically splice like a cis intein.

While the construct expressed at the right size, we did not observe band formation corresponding to the spliced product.

On this gel we also tested diafiltration of pVS30 (E=elution, Df=after buffer exchange) to ensure our protein was retained during buffer exchange.

pHG68 expression test (SDS-PAGE)

Mon (10/7/2019) Large scale expression of pHG(44,52,60,64)

Grew 200mL cultures of pHG(44,52,60,64) to OD600=0.8-0.9 and induced with 1mM IPTG for 4 hours. Cultures were pelleted down and stored in -80 C for purification.

Mon (10/7/2019) Test diafiltration of pVS29

Tested the new diafiltration columns on pVS29 to concentrate and exchange buffers.

Wed (10/9/2019) Expression of pVS47

Large scale expression of pVS47 for lysate mixture tests.

Wed (10/9/2019) Gibson assemblies

Redid Gibson assemblies of constructs that failed colony PCR screening from the third nested intein set and the split-linker set.

Wed (10/9/2019) Purification of pHG(44,52,60,64)

Purified 200mL cultures of pHG(44,52,60,64) from frozen pellets.

All contructs except for pHG60 purified well.

pHG44 also has a relatively thick band in the elution below the expected size which we believe is a degredation product containing maltose binding protein.

U=uninduced, I=induced, P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

Purification gel (SDS-PAGE)

Purification gel (SDS-PAGE)

Wed-Fri (10/9/2019)-(10/11/2019) Expression, purification and diafiltration of pVS48
Ripal, Jemy, Mel

Large scale expression of pVS48 followed by purification and diafiltration.

Thu (10/10/2019) Diafiltration of pHG(44,52,60,64)

Elutions of the four constructs were concentrated in centrifugal diafiltration columns and resuspended in a splicing buffer with high glycerol content intened to mimick the intracellular environment.

Thu (10/10/2019) In-vitro assay screen

Several of the constructs were mixed in pairwise in-vitro assays to see if splicing would occur.

We were able to observe splicing between pHG44 and pHG64 however the concentration of pHG52 and pHG60 was too low to show up clearly on a gel.

Splicing assay (SDS-PAGE)

Important reactants and products are annotated

Splicing assay (SDS-PAGE)

Splicing assay (SDS-PAGE)

Fri (9/11/2019) Diafiltraion of pVS31
Mel, Ripal
Sat (10/12/2019) Large scale expression of pHG(44,59,60,68)

Grew 200mL cultures of pHG(44,59,60,68) to OD600=0.7-0.8 and induced with 1mM IPTG at room temperature for 4 hours.

Cells were pelleted and stored in -80 C for purification.

Sat (10/12/2019) In-vitro kinetic measurements

After observing splicing between pHG44 and pHG64, we ran another gel and took aliquots at certain time intervals to develop an understanding of the reaction rate.

We also ran both starting constructs at t=4 hours as negative controls to ensure we were not seeing degredation products.

Splicing kinetic assay (SDS-PAGE)

Important reactants and products are annotated

Sun (10/13/2019) Transformation and colony PCR of BBa_K590016

Transformed a construct from the 2019 iGEM distribution kit for characterization.

Week 23

Mon (10/14/2019) Expression of pVS47 and pVS48

Large scale expression of pVS47 and pVS48 for lysate mixture tests.

Mon (10/14/2019) Purification of pHG(44,59,60,68)

Purified 200mL cultures of pHG(44,59,60,68). All constructs purified well.

U=uninduced, I=induced, P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

Purification gel (SDS-PAGE)

Purification gel (SDS-PAGE)

Tue (10/15/2019) Expression of pVS45 and pVS29

Large scale expression of pVS45 and pVS29 for lysate mixture tests.

Tue (10/15/2019) In-vitro screening of new constructs

Performed overnight in-vitro assays of several combinations of the recently purified constructs.

We observed splicing between pHG44 and pHG64 (as seen before) and also a hint of splicing between pHG60 and pHG64 (predicted).

However, we did not observe splicing between pHG59 and pHG44 as expected. After carefully reviewing our construct sequences, we realized that our design was missing a single amino acid residue in gp41-1 N.

In-vitro assay screening (SDS-PAGE)

The four constructs on the left are at t=0

For each reaction mixture, the left lane is t=0 and the right lane is t=20 hours at 37 C