iGEM 2018 — Team Hawaii

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# Team:Hawaii/Notebook

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  + [CALENDAR](https://2018.igem.org/Team%3AHawaii/Calendar)

* May 14 - May 18
* [May 21 - May 25](#week1)
* [May 28 - Jun 1](#week2)
* Jun 4 - Jun 8
* Jun 11 - Jun 15
* Jun 18 - Jun 22
* Jun 25 - Jun 29
* Jul 2 - Jul 6
* Jul 9 - Jul 13
* Jul 16 - Jul 20
* Jul 23 - Jul 27
* Jul 30 - Aug 3
* Aug 6 - Aug 10
* Aug 13 - Aug 17
* Aug 20 - Aug 24
* Aug 27 - Aug 31
* Sep 3 - Sep 7
* Sep 10 - Sep 14
* Sep 17 - Sep 21
* Sep 24 - Sep 28
* Oct 1 - Oct 5
* Oct 8 - Oct 12
* Oct 15 - Oct 19

# MAY 14 - MAY 18

Bacteria media, milli Q water, buffers and LB agar plates were restocked in preparation for the upcoming intensity of our lab activities. While our undergraduates were preparing for final exams, iGEM graduate student Ryan designed and ordered the CR2 Gag-Protease-RT construct for our first experiment - determining the cleavage sites of the aspartyl-like protease.

## HIGHLIGHTS

* [Designing the CR2 Gag-Protease-RT construct](#subtitle1)
* [Diagnostic digest and plasmid extraction](#subtitle2)

### Designing the CR2 Gag-Protease-RT construct

The CR2 Gag-Protease-RT construct consists of a consensus sequence produced from 171 sequences of this Zea mays centromere retrotransposon (CR) subfamily and includes the conserved gag, protease, and reverse transcriptase domains (Conserved Domain Basic Local Alignment Search Tool). The sequences were gathered and assembled by Dan Laspisa. The construct contains N-terminal and C-terminal 8-histidine tags followed by a TEV cleavage site for later nickel resin purification and was cloned into the pET14b vector (GenScript) using NcoI and BamHI. Ryan performed transformations into DH5-alpha and BL21 cells for long term plasmid storage and expression respectively.

| CR2 Gag-Protease-RT Construct | |
| --- | --- |
| Amplicon Size (bp) | 2342 |
| Protein Size (kDa) | 90 |
| 5' RE Site | NcoI |
| 3' RE Site | BamHI |
| Purification | C + N-terminal 8-His and TEV cleavage site |
| Vector | pET14b |

### Diagnostic digest and plasmid extraction

A restriction enzyme digest of our CR2 Gag-Protease-RT construct was conducted to confirm plasmid uptake for two of our colonies. A plasmid extraction of the DH5-alpha colonies with this insert and a BL21(DE3) pLysSs transformation was performed.

# MAY 21 - MAY 25

Colony PCR reactions and gel runs were performed throughout the week to confirm the presence of our insert in DH5-alpha and BL21 cells. Plasmids were extracted for sequence verification and a small scale 4 hour induction was performed on our BL21 cells that revealed successful expression of the full polyprotein at 28C. Meanwhile, Gina is starting to work on the Wiki and the team is researching optimized buffer conditions for aspartyl proteases in preparation for dialysis after purification.

## Highlights

* [Confirming the presence of our CR2 Gag-Protease-RT insert](#subtitle4)
* [Verifying the sequence of the CR2 Gag-Protease-RT insert](#subtitle5)
* [Expression of the full CR2 Gag-Protease-RT polyprotein at 28C](#subtitle6)

### Confirming the presence of our CR2 Gag-Protease-RT insert

A colony PCR of the DH5a CR2 Gag-Protease-RT construct containing cells initially performed by Ryan revealed a puzzling gel image. Emily and Gina performed a colony PCR of the BL21 CR2 Gag-Protease-RT construct containing cells and ran it on a 1.5% agarose gel. However, there were still no indicative bands of the insert. Emily and Gina then conducted another set of PCR reactions with leftover bacteria in the PCR tubes (invisible to the eye) in thousand-fold and million-fold dilutions and modified the PCR parameters. The gel run this time revealed our insert band at ~2 kb.

| Colony PCR Reaction Mixture | |
| --- | --- |
| Reagent | Amount (ul) |
| Template | 11 ul |
| 5X OneTaq Buffer | 3 ul |
| dNTP Mix | 0.3 ul |
| T7 Forward | 0.3 ul |
| T7 Reverse | OneTaq |
| Total | 15 ul |

| Parameters for Colony PCR | | | |
| --- | --- | --- | --- |
| Initial PCR Parameters | | Modified PCR Parameters | |
| Temperature (C) | Time | Temperature (C) | Time |
| 94 | 3 min. | 94 | 3 min. |
| 94 | 20 sec. | 94 | 20 sec. |
| 65 | 20 sec | **\*50** | 20 sec |
| 68 | 1 min. 10 sec. | 68 | **\*3 min.** |

### Verifying the sequence of the CR2 Gag-Protease-RT insert

A plasmid extraction was performed by Shelby, Fernanda, and John from CR2 Gag-Protease-RT BL21 cells that were grown overnight to send to sequencing with primers flanking our insert and internal primers. The sequencing machine was expected to yield ~500-800 readable bases, thus, internal primers were ordered to cover the gaps. Initially, an incorrectly labelled buffer was added during plasmid extraction so DNA concentration was low (5 ng/ ul). However, there was enough supernatant to try it again. After Nanodrop Quantification, a much higher DNA concentration (70-90 ng/ul) was obtained. PCR tubes were then prepared with DNA, primers (T7F, T7R, Internal GagF, Internal GagR), and PCR grade H20 and submitted it to the sequencing lab on campus. We set up overnight cultures of colonies 1, 2, and 3 for induction the next morning.

### Expression of the full CR2 Gag-Protease-RT polyprotein at 28C

A 4-hour induction at 28C or 37C with 0.5 mM or 1 mM IPTG was performed by Gina and later run on a 15% SDS-PAGE gel by Emily. The 28C gel revealed higher intensity bands slightly under the 100 kDa ladder band which corresponded to the ~90 kDa full polyprotein. At 37C, little to no polyprotein expression could be differentiated between uninduced and induced cells.

# MAY 28 - JUN 1

Bands corresponding to partial degradation fragments after protease cleavage were extracted from 15% SDS-PAGE gels and sent to a mass spectrometry facility to reveal protease cut sites. A soluble purification was performed on the induced BL21 cells and all fractions (total protein, wash, and elution) were run on a 15% SDS-PAGE gel.

## Highlights

* [Analysis of polyprotein expression at 28C, 32C, and 35C](#subtitle7)
* [Soluble purification confirms absence of polyprotein in soluble fraction](#subtitle8)
* [Sending bands corresponding to our polyprotein fragments to Taplin](#subtitle9)

### Analysis of polyprotein expression at 28C, 32C, and 35C

To determine the effect of temperature on protease activity, a 6 hour induction was performed at 32C or 35C. Induced cells were run on a 15% SDS-PAGE gel at 70 V for stacking and 100 V for the resolving gel. A gel was run with cells from all different temperatures so far, however, unclear separation in the gel prevented us from coming to a definite conclusion about the effect of temperature on protease activity.

### Soluble purification confirms absence of polyprotein in soluble fraction

Although we expected to find our polyprotein in the insoluble fraction, a soluble purification of induced cells were performed to confirm this. During soluble purification, cells were nutated in wash (Lysis Equilibrium Wash buffer) and elution buffers. All fractions were run on a 15% SDS-PAGE gel.

### Sending bands corresponding to our polyprotein fragments to Taplin

~90 kDa, ~75 kDa, and ~50 kDa bands were extracted from three different 15% SDS-PAGE gels containing 6 hour induced BL21 cells at 32 C and sent to the Taplin Mass Spectrometry Facility to reveal protease cut sites. Ryan and Vishal analyzed bands to extract from and conducted the gel extraction.

# JUN 4 - JUN 8

We explored our iGEM Distribution Kit and transformed E. coli to express red fluorescent protein (RFP). Various insoluble protein purifications were performed on our induced CR2 Gag-Protease-RT containing cells and run on 15% SDS-PAGE gels.

## Highlights

* [Expression of RFP in E.coli](#subtitle10)
* [Insoluble purification reveals CR2 Gag-Protease-RT polyprotein in inclusion bodies](#subtitle11)

### Expression of RFP in E.coli

Shelby performed a transformation of E.coli to express RFP using the guides provided by iGEM. The RFP BioBrick from the iGEM kit plate #3 (part BBa\_E1010) was used. The first transformation using previous transformation protocols from our lab notebook did not yield visible colonies. However, the second transformation using transformation protocols specified by iGEM yielded red colonies in the control plate. Shelby plans to make a working stock of transformed E.coli using the positive control colonies.

### Insoluble purification reveals CR2 Gag-Protease-RT polyprotein in inclusion bodies

Gina and Jon performed an insoluble protein purification of CR2 Gag-Protease-RT containing cells using nickel-resin, Lysis Equilibrium Wash buffer, and elution buffer. A 15% SDS-PAGE gel revealed ~90 kDa, ~75 kDa, and ~50 kDa fragments corresponding to the expected degradation fragments from our polyprotein. An insoluble protein purification without lysozyme was performed by Gina as lysozyme was observed to have a similar molecular weight to the protease. The fractions of this purification were run on a tricine gel in the hopes of being able to observe the smaller ~10-23 kDa fragments. However, this was the first time our lab performed a tricine gel run and experimental protocols were used.
The Tricine gel experiment was unsuccessful in separating the protease and the smaller RT protein. This may have been due to the modifications made to Haider et al., 2012’s protocols, such as running the gel at 70 V stacking and 150 V running rather than the continuous 125-150 V. We decided that we will determine the RT and protease sequence via mass spectrometry and trypsin digest rather than redoing another tricine gel experiment.

# JUN 11 - JUN 15

A 20C induction was performed to further test protease activity at various temperatures and cells induced at 20C, 28C, and 35C were run on the same 15% SDS-PAGE gel. Attempts at verifying the sequence of our insert and Shelby’s RFP project are continued.

## Highlights

* [Inducing cultures at 20C, 28C, and 35C to determine the effect of temperature on protease activity](#subtitle12)
* [Sequence verification attempts of BL21 and DH5-alpha cells](#subtitle13)
* [Colony PCR of transformed E.coli with RFP](#subtitle14)

### Inducing cultures at 20C, 28C, and 35C to determine the effect of temperature on protease activity

A 6 hour induction of our CR2 Gag-Protease-RT BL21 cells at 20C was performed to investigate the effect of even lower temperatures on protease activity. A 6 hour induction of cells at 28C and 35C was performed a few days later to run all induced and uninduced cells at 3 different temperatures. Unfortunately the bands did not separate well but slightly show partial degradation products at all induced temperatures. The gel will be rerun next week.

### Sequence verification attempts of BL21 and DH5-alpha cells

Our previous internal primers could not completely cover the full length CR2 Gag-Protease-RT sequence. New internal primers were ordered to sequence our entire ~2 kb insert and used in our sequencing reactions. Initial overnight cultures of DH5-alpha yielded low concentration of DNA, thus, another plasmid prep was run the next day. Plasmid extraction of BL21 cells were sent to sequencing with the new primers. Unfortunately, chromatogram results did not yield the ~500-800 bp expected from the sequencing machine. In order to continue the team’s efforts and ensure better results, Jon took on the task of verifying sequences and will investigate the reason for our poor sequencing results.

### Colony PCR of transformed E.coli with RFP

A colony PCR to verify the insert was performed with transformed E.coli expressing the RFP.

| Reaction Mix (1 Reaction) | |
| --- | --- |
| Reagent | Amount (ul) |
| Template | 11 |
| 5X OneTaq Buffer | 3 |
| dNTP mix | 0.3 |
| VF2 Primer | 0.3 |
| VR Primer | 0.3 |
| OneTaq | 0.1 |
| Total | 15 |

| Parameters for Colony PCR (35 cycles) | |
| --- | --- |
| Temperature (C) | Time |
| 94 | 3 min. |
| 94 | 20 sec. |
| 55 | 20 sec. |
| 68 | 45 sec. |
| 68 | 5 min. |
| 4 | Infinite (Storage) |

# JUN 18 - JUN 22

The 20C, 28C, and 35C gels from last week was rerun to obtain a better resolution and a PCR was conducted off of leftover plasmid to send in for another attempt at sequencing. Shelby continues her work on the RFP transformed cells and starts overnight cultures.

## Highlights

* [Analysis of protease activity at 20C, 28C, and 35C](#subtitle15)
* [PCR of DH5a plasmids to amplify insert for later sequencing](#subtitle16)

### Analysis of protease activity at 20C, 28C, and 35C

Another SDS-PAGE gel was made to run induced cultures from last week. The resolution obtained was much better, possibly meaning that the person who made the gel last week may have added incorrect reagent amounts. In this gel, a ~50 kDa band appears to be present in 20C and 28C induced cultures, while a stronger ~90 kDa band is observed in the 28C induced cultures. At 35C, there seems to be no difference between induced and uninduced cultures. This may be due to nonspecific cleavage or higher activity of the protease at this temperature.

### PCR of DH5a plasmids to amplify insert for later sequencing

PCR and PCR cleanup (QIA-quick PCR purification protocol) was run to perform sequencing reactions until the insert sequence can be successfully verified. 250 ng of DNA was used for each reaction.

| # | Template | DNA conc. (ng/ul) |
| --- | --- | --- |
| 1 | CR2 Gag-Protease-RT (DH5a) colony 1 | 97.1 |
| 2 | CR2 Gag-Protease-RT (DH5a) colony 2 | 49.6 |

| PCR Reaction Mixture | | |
| --- | --- | --- |
| Template | 2.57 | 5.04 |
| 5X OneTaq Buffer | 10 | 10 |
| 10 mM dNTP | 1 | 1 |
| 10 mM T7 Forward | 1 | 1 |
| 10 mM T7 Reverse | 1 | 1 |
| PCR grade H2O | 34.18 | 31.71 |
| Total | 50 | 50 |

| PCR Thermocycle Parameters | |
| --- | --- |
| 94 | 1 min. |
| 94 | 20 sec. |
| 50 | 20 sec. |
| 68 | 3 min. |
| 68 | 5 min. |
| 4 | Infinite (Storage) |

# JUN 25 - JUN 29

Mass spectrometry results came in from Taplin! Fragments were annotated on the sequence file and revealed a protease cleavage site downstream of the nucleocapsid. Unfortunately, we were unable to identify the second protease cleavage site through this data. However, the team decided to move forward into the next major experiment - determining the presence of VLPs in various polyproteins. These polyproteins would consist of combinations of the domains with both determined and putative protease cut sites. The team is beginning to seriously consider dynamic light scattering, split proteins, and other equipment for VLP assessment and debated Gibson vs. Biobrick for the initial assembly of parts. Jon and Fernanda sent the PCR products to sequencing and had a discussion with the sequencer to investigate why sequencing chromatograms were not coming out as expected.

## Highlights

* [Mass spectrometry results reveal protease cleavage site downstream of the nucleocapsid](#subtitle17)
* [Discussing combinatorial assay for VLP formation](#subtitle18)
* [Analysis of previous teams working with virus like particles (VLP)](#subtitle19)

### Mass spectrometry results reveal protease cleavage site downstream of the nucleocapsid

Tryptic fragments for each band were mapped onto the CR2 Gag-Protease-RT GenScript construct using fragment data copied from the Taplin results page into an Excel sheet and sorted by position. The 100 kDa fragments covered 90.04 kDa, the 75 kDa fragments covered 56.76 kDa, and the 50 kDa fragments covered 47.88 kDa of the full construct. These fragments confirmed a protease cleavage site downstream of the nucleocapsid in the form of an 18 bp gap between the end of the last 50 kDa fragment (6 bp upstream of the 3’ end of the CCHC Zinc Finger domain) and the beginning of the next 75 kDa and 100 kDa fragments (12bp after the 3’ end of the CCHC Zinc Finger). The second cleavage site between the protease and reverse transcriptase domains could not be identified as the 75 kDa fragments did not exceed 56.76 kDa, with the most upstream protease cut site observed being located well within the protease domain. This possibly suggests that either the wrong band was excised from the PAGE gel or that the protease has auto digestive activity under the conditions used.

### Discussing combinatorial assay for VLP formation

With only one cut site confirmed from the mass spectrometry results, the team decided to proceed with expressing variations of our CR2 Gag-Protease-RT construct with differences in presence and order of the domains, purification tags, and/or reporter sequences. This week, the team researched possible split reporter proteins and conducted a literature search on potential colorimetric assays.

### Analysis of previous teams working with virus like particles (VLP)

The team conducted a quick search on previous iGEM teams that worked with VLPs. We found great resources from the 2012 Wageningen Wiki with VLP assembly and disassembly protocols. While surveying other teams, we also stumbled on split reporter parts from the 2017 Wageningen Wiki and sent an inquiry to Abigail, our iGEM HQ representative, about how we could obtain the parts.

### Gibson vs. Biobrick assembly considerations

After considering what the combinatorial polyprotein assay would entail, the team decided to proceed with Gibson assembly to put together the initial parts. Afterwards, we would clone the successful parts into a Biobrick for submittal.

### Investigation of unsatisfactory sequencing results reveal too much DNA as the cause

Following Jon and Fernanda’s discussion with the sequencer on campus, they re-submitted one tube with less DNA per his suggestion. The sequencing results came out much better! They then diluted and prepared the rest of the tubes for submittal. These results allowed us to confirm that we had the correct insert sequence in our DH5a colony.

# JUL 2 - JUL 6

A same day induction at 20C, 28C, and 35C with subsequent 15% SDS-PAGE gel run confirmed protease activities at different temperatures. The team finalized the initial part combinations and ordered the first set of primers (and Gibson primers) for PCR amplification and assembly next week. In addition, the team surveyed all 342 team wikis to discover any teams with similar interests to collaborate on.

## Highlights

* [Confirmation of protease activities at 20C, 28C, and 35C](#subtitle22)
* [Wiki Survey](#subtitle23)
* [Polyprotein combinations and primer ordering](#subtitle24)

### Confirmation of protease activities at 20C, 28C, and 35C

Previous induction and gel runs for the protease experiment were performed in parts by various team members on different days of the week. This may have contributed to our unclear results on the effect of induction temperature on protease activity. To eliminate these variables, a 200 mL 20 C, 28 C, and 35 C induction and same day SDS-PAGE run was performed by one team member. Dr. Robert Paull graciously granted the team access to a third shaking incubator to perform our three inductions all at the same time. Three autoclaved 500 mL flasks covered with layers of aluminum foil were used for induction of the 200 mL cultures and 50 mL falcon tubes were used to grow the uninduced cells. The bands visible on the 15% SDS-PAGE gel conducted on the same day of the induction confirms that 20C is best for producing the full length gag protein (50 kDa), 28C is best for producing the full length CR2 Gag-Protease-RT polyprotein and 35C produces less protein than at any other temperature we have induced with so far.

### Wiki Survey

The team divided the 342 team wikis into ~50 wikis to scan per person. We found that some teams were working on nanocompartments and using capsid proteins, but no other team was working with retrotransposons or looking to express virus-like particles (VLPs). Some teams were thinking of using split reporter systems, however, many of the descriptions were still slightly vague. Although we were not able to find anything extremely similar to our own project, It was interesting to see all the different ideas that other teams came up with to tackle their problem.

### Polyprotein combinations and primer ordering

After filtering through a list of all possible combinations of domains, purification tags, and reporter sequences that would make up our polyprotein, the team decided on an initial list of polyproteins to assemble. We decided to amplify this off of the existing CR2 Gag-Protease-RT construct. Thus, C-terminal purification tag constructs would need to be amplified in fragments, then ligated through Gibson Assembly. We then spent an afternoon together creating regular and Gibson primers for PCR amplification of our pieces and subsequent Gibson assembly next week.

| List of Initial Polyprotein Constructs | | |
| --- | --- | --- |
| 1 | Construct | Amplicon (bp) |
| 2 | HisTEV-CA-NC | ~1300 |
| 3 | HisTEV-CA1 | ~950 |
| 4 | HisTEV-CA2 | ~1100 |
| 5 | CA-NC-TEVHis | ~1300 |
| 6 | CA1-TEVHis | ~950 |
| 7 | CA2-TEVHis | ~1100 |

# Jul 9 - Jul 13

In preparation for our polyprotein assembly, we cloned and digested the pET14b plasmid and amplified the parts using primers. A 1.5% agarose gel confirmed the sizes of our amplicons.

## Highlights

* [Digesting the pET14b plasmid with NcoI and BamHI](#subtitle25)
* [Amplifying polyprotein pieces](#subtitle26)
* [Growing DH5a with the Gag-Prot-RT plasmid](#subtitle27)

### Cloning and digesting the pET14b plasmid

pET14b was obtained from a glycerol stock of transformed DH5-a cells. A 100 ml overnight culture was made, and a maxi-prep was done to isolate the plasmid. A double digest was then conducted using the NcoI and BamHI restriction enzymes and a gel was run on the both the whole and digested plasmid to confirm that the plasmid was cut and linearized. Our linearized plasmid showed a band size of about 8-10 kb, which was larger than expected (pET14b is 4.6 kb).

### Amplifying polyprotein pieces

A PCR was conducted to amplify all of our construct fragments. For C-terminal purification tag constructs, two fragments were amplified: one larger fragment that included the capsid (~900-1000 bp), and a smaller fragment that included the TEV cleavage site and histidine tags (~60 bp).

| PCR Reaction Mix | |
| --- | --- |
| Reagent | Amount (ul) (1 Reaction) |
| 5X OneTaq Buffer | 9 |
| dNTP mix | 0.9 |
| OneTaq | 0.1 |
| H2O | 30 |
| Template (diluted 0.42 ng/ul) | 1 |
| Total Volume | 41 |

| Primers Added to Each Tube | |
| --- | --- |
| Respective Forward primer | 0.9 |
| Respective Reverse primer | 0.9 |

| PCR Thermocycle Parameters (Larger fragments) | |
| --- | --- |
| 94 | 3 min. |
| 94 | 20 sec. |
| 61 | 20 sec. |
| 68 | 1 min. 15 sec. |
| 68 | 5 min. |
| 4 | Infinite (Storage) |

| PCR Thermocycle Parameters (Smaller fragments) | |
| --- | --- |
| 94 | 3 min. |
| 94 | 20 sec. |
| 60 | 20 sec. |
| 68 | 20 sec. |
| 68 | 5 min. |
| 4 | Infinite (Storage) |

### Growing DH5a with the Gag-Prot-RT plasmid

A 100ml overnight culture was done on transformed DH5-a cells containing the Gag-Prot-RT construct (GPR). A maxi-prep was done on the culture to isolate and purify the plasmid. A gel was done on the purified GPR construct with the plasmid to confirm plasmid presence. Nanodrop quantification revealed a very low yield from the first maxi-prep (~12 ng/µl) in our 20 µl sample, so a second overnight culture was prepared and a second maxi prep was done. The second maxi-prep yielded a 20 µl sample that was 136 ng/µl.

# JUL 16 - JUL 20

Bacterial cultures containing the CR2 Gag-Prot-RT plasmid were grown overnight from our DH5a glycerol stocks. The pET14b plasmids and PCR amplicons from last week were digested, cleaned, then ligated. Multiple ligation reactions were created with varying amounts of vector. Transformations into commercial and homemade DH5a and BL21 cells were conducted later in the week. The second transformation initially appeared to be successful.

## Highlights

* [Gel extraction and EtOH precipitation of vector](#subtitle28)
* [RE digestion and MinElute Cleanup](#subtitle29)
* [Ligation reactions with varying amounts of vector](#subtitle30)
* [Transformation in DH5a and BL21 with constructs 1, 2, 3](#subtitle31)

### Gel extraction and EtOH precipitation of vector

In order to obtain more pET14b vectors, we grew overnight cultures of bacteria containing pET14b plasmids with our CR2 Gag-Protease-RT insert, digested them with NcoI and BamHI, then conducted a gel extraction of the 4671 bp vector. Following EtOH precipitation and Nanodrop Quantification, we were able to use the vector for our ligation reactions and yielded enough vector for ~30 more ligation or Gibson Assembly reactions.

### RE digestion and MinElute Cleanup of PCR amplicons

PCR amplicons from last week and empty pET14b plasmids were digested with NcoI and BamHI primers, then cleaned using the MinElute cleanup kit. After cleanup, we were able to ligate these to our pET14b vector and store them at -4C overnight for transformation the next day.

### Ligation reactions with varying amounts of vector

We conducted 3 sets of ligation reactions this week with different amounts of vector in a 1:3 vector:insert molar ratio. We ligated partial Gibson Assembly fragments to the vector in preparation for Gibson Assembly next week.

| Ligation Reaction Mix #1 for Constructs 1 - 5a | | | | | |
| --- | --- | --- | --- | --- | --- |
| Reagent | 1 (ul) | 2 (ul) | 3 (ul) | 4a (ul) | 5a (ul) |
| PCR grade H2O | 5.5 | 5.5 | 5.5 | 5.5 | 5.5 |
| 10X ligation buffer | 1 | 1 | 1 | 1 | 1 |
| Vector (8.4 ng) | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
| Insert | 1 | 1 | 1 | 1 | 1 |
| T4 DNA Ligase | 1 | 1 | 1 | 1 | 1 |
| Total | 10 | 10 | 10 | 10 | 10 |

| Ligation Reaction Mix #2 for Constructs 1 - 5a | | | | | |
| --- | --- | --- | --- | --- | --- |
| Reagent | 1 (ul) | 2 (ul) | 3 (ul) | 4a (ul) | 5a (ul) |
| PCR grade H2O | 5.39 | 6.17 | 5.73 | 4.11 | 5.68 |
| 10X ligation buffer | 1 | 1 | 1 | 1 | 1 |
| Vector (21.5 ng) | 1 | 1 | 1 | 1 | 1 |
| Insert | 1.61 | 0.83 | 1.27 | 2.89 | 1.32 |
| T4 DNA Ligase | 1 | 1 | 1 | 1 | 1 |
| Total | 10 | 10 | 10 | 10 | 10 |

| Ligation Reaction Mix #3 for Constructs 1 - 3 | | | |
| --- | --- | --- | --- |
| Reagent | 1 (ul) | 2 (ul) | 3 (ul) |
| PCR grade H2O | 3.69 | 5.20 | 4.39 |
| 10X ligation buffer | 1 | 1 | 1 |
| Vector (44.1 ng) | 1 | 1 | 1 |
| Insert | 3.31 | 1.80 | 2.61 |
| T4 DNA Ligase | 1 | 1 | 1 |
| Total | 10 | 10 | 10 |

### Transformation in DH5a and BL21 with constructs 1, 2, 3

We conducted one set of transformations in DH5a and BL21 and another of DH5a on both commercial and homemade competent cells. The first transformation used ligation reactions with 8.4 ng of vector and the second transformation used ligation reactions with 21.5 ng of vector. Our observations were as follows.

**Transformation 1 - BL21 and DH5a with ligation reactions containing 8.4 ng of vector**

* 2 large white colonies on positive control plate with commercial BL21 cells
* Numerous small white scattered colonies on positive control plate with commercial DH5a cells
* Few white colonies with smaller satellite colonies on positive control plate with homemade DH5a cells
* No colony growth on negative controls

**Conclusions**

* Plates contained the correct antibiotics
* Homemade DH5a cells are competent, but much less efficient than commercial cells
* 8.4 ng may be too little vector to ligate with

**Transformation 2 - DH5a with ligation reactions containing 21.5 ng of vector**

* ~105 small white colonies scattered across the commercial DH5a plate with part 1
* ~135 small white colonies scattered across the commercial DH5a plate with part 2
* ~100 small white colonies scattered across the commercial DH5a plate with part 3
* ~5-7 small white colonies in a cluster on the homemade DH5a plate with part 1
* ~5-7 small white colonies in a cluster on the homemade DH5a plate with part 2
* ~5-7 small white colonies in a cluster on the homemade DH5a plate with part 3

**Conclusions**

* 21.5 ng of vector in ligation reactions seem to be enough to yield colonies scattered across the plates of competent cells

| Transformation for DH5a (H=homemade, C=commercial) | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Reagents | 1 (H) (ul) | 2 (H) (ul) | 3 (H) (ul) | 1 (C) (ul) | 2 (C) (ul) | 3 (C) (ul) | (+) (ul) | (-) (ul) |
| DH5a Competent Cells | 40 | 40 | 40 | 40 | 40 | 40 | 40 | 40 |
| pUC19 | - | - | - | - | - | - | 5 | - |
| Ligated Vector (2 fold TE dilution) | 5 | 5 | 5 | 5 | 5 | 5 | - | - |
| Total | 45 | 45 | 45 | 45 | 45 | 45 | 45 | 40 |

| Transformation for BL21 (H=homemade, C=commercial) | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Reagents | 1 (H) (ul) | 2 (H) (ul) | 3 (H) (ul) | 1 (C) (ul) | 2 (C) (ul) | 3 (C) (ul) | (+) (ul) | (-) (ul) |
| Bl21 Competent Cells | 40 | 40 | 40 | 40 | 40 | 40 | 40 | 40 |
| 23 mM B-ME | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| pUC19 | - | - | - | - | - | - | 5 | - |
| Ligated Vector (2 fold TE dilution) | 5 | 5 | 5 | 5 | 5 | 5 | - | - |
| Total | 46 | 46 | 46 | 46 | 46 | 46 | 46 | 41 |

# JUL 23 - JUL 27

Colony PCR and sequencing revealed that our transformation was unsuccessful and steps throughout the way may have introduced contaminants.

## Highlights

* [Colony PCR of DH5α transformants with constructs 1, 2, 3](#subtitle31)
* [Sequencing of DH5a transformants reveal incorrect bacterial insert](#subtitle32)

### Colony PCR of DH5α transformants with constructs 1, 2, 3

A colony PCR was run to verify that colonies contain correct insert sizes for constructs 1, 2, and 3. The first 8 colonies were run on a 1.5% agarose gel. Only one colony per construct seemed to contain an insert ~200-300 bp lower than the expected size. The gel appeared to contain aberrations that may have contributed to the unexpected result. Colonies containing our possible insert were grown overnight for sequencing the next morning.

### Sequencing of DH5a transformants reveal incorrect bacterial insert

A protein BLAST search of the sequences received for the colonies grown reveal a bacterial insert. The top hits were Beta-lactamase and ATP synthase for E.coli. This suggests that our ligation reactions may have been contaminated with the original insert present in the empty vector which may not have been cleaned with our spin columns.

# JUL 30 - AUG 3

Several attempts of PCRs were made to obtain amplicons corresponding to constructs 1, 2, and 3. However, we consistently received no yield of amplicon 1 despite previously having success with the same thermocycler parameters and reagent amounts. Throughout the week, we tested different parameters and finally saw better yield when using OneTaq GC Reaction Buffer and GC Enhancer. While these GC enhancements did produce amplicon 1, the production of amplicons 2 and 3 seemed less enhanced with these reagents. A successful transformation with construct 2 was also conducted and the colony was sequence verified by the end of the week.

## Highlights

* [PCR with and without GC enhancements](#subtitle33)
* [First successfully transformed DH5a cells with construct 2](#subtitle34)

### PCR with and without GC enhancements

The use of GC Buffer instead of regular Reaction buffer seemed to help produce stronger bands for our high GC content (55-60%) amplicons.

### First successfully transformed DH5a cells with construct 2

We obtained 1 colony that contained the correct insert following Colony PCR and sequencing. This colony was then grown overnight and transformed into BL21.

# AUG 6 - AUG 10

One N-terminal purification tagged construct is pushed forward and placed in BL21 for induction. Due to our unsuccessful transformations, we investigated possible reasons for our poor results. In addition, Gibson Assembly for the formation of our C-terminal tagged constructs is tested.

## Highlights

* [Transformation and Induction of BL21 cells with construct 2](#subtitle35)
* [Optimizing heat shock for homemade competent cells](#subtitle36)
* [Protein purification of BL21 cells with construct 2](#subtitle37)
* [Gibson Assembly of C-terminal purification tag constructs](#subtitle38)
* [2-Step PCR of C-terminal purification tag constructs](#subtitle39)

### Transformation and Induction of BL21 cells with construct 2

A sequence verified BL21 colony with our construct 2 insert was induced at 20C and 28C with 0.5 mM IPTG. At both temperatures, the 37 kDa protein was successfully expressed. However, at 28C, much more of our protein was produced. A large scale 100 mL induction at 28C was performed the next day for subsequent protein purification.

### Optimizing heat shock for homemade competent cells

Three transformations of DH5a were conducted using the same amount of plasmid, but with varying heat shock durations at 20 seconds, 30 seconds, and 45 seconds. Only 2 colonies were present with the 45 second transformation, while 100+ colonies appeared on the 30 second and 40+ colonies appeared on the 20 second plate. These findings suggest that our transformations may have yielded so few colonies due to our use of the 45 second heat shock specified in our protocol.

### Protein purification of BL21 cells with construct 2

Soluble and insoluble fractions of the induced cultures run on a SDS PAGE gel revealed that the protein was in the insoluble fraction. An insoluble purification with 700 mM NaCl and 30 mM imidazole washes was conducted and concentrated using the Pierce protein concentrator.

### Gibson Assembly of C-terminal purification tag constructs

A Gibson Assembly of C-terminal purification tagged constructs was conducted for 15 minutes and 60 minutes with the positive control (NEBuilder) provided. The samples were run on a 1.5% agarose gel as the manual indicated that an efficient reaction will show assembled products of the correct size and disappearance of fragments. However, the larger of the two fragments that was supposed to be assembled was observed on the gel. The positive control did not show up on a gel, however, the reaction consisted of all commercially obtained reagents. We decided to contact NEB and inquire about our results next week.

### 2-Step PCR of C-terminal purification tag constructs

A 2-step PCR was conducted to add on the C-terminal TEV-8xHis tag. Amplicons were extracted from a low melt gel, digested, cleaned, then run on another agarose gel to confirm the amount of DNA present in each extract.

# AUG 13 - AUG 17

Additional modifications were made to our protocols to increase our transformation yield. Our lack of results after running the Gibson Assembly sample on an agarose gel lead us to contact the supplier.

## Highlights

* [New findings about Gibson Assembly after speaking with NEB](#subtitle40)
* [Gibson Assembly confirmation through transformation into DH5a](#subtitle41)
* [Optimizing ligation and transformation](#subtitle42)

### New findings about Gibson Assembly after speaking with NEB

After speaking with a product manager at NEB, we found that the positive control provided for Gibson Assembly would not necessarily show up if run on a gel. Instead, we would have to transform it to confirm the success of our assembly.

### Gibson Assembly confirmation through transformation into DH5a

A transformation was conducted with the Gibson samples, however, no colonies were observed on all plates. This may be due to the transformation protocol itself rather than the Gibson Assembly. Another transformation using modified transformation procedures below was conducted, yet still yielded no colonies.

### Optimizing ligation and transformation

Throughout the week we faced difficulties in obtaining colonies when transforming into our homemade DH5a competent cells, even after optimization of the heat shock duration. We tried the following new techniques which seemed to produce more colonies.

Ligation

* Run insert samples on an agarose gel to estimate DNA amount instead of using Nanodrop Quantification
* Conduct a "melting ice" ligation

Transformation

* Thaw competent cells on ice
* Use 1 ul of 23 mM beta-mercaptoethanol
* 30 second heat shock instead of 45 seconds

# AUG 20 - AUG 24

The optimized protocols allowed us to transform all N-terminal purification tagged constructs. However, efforts were still being made to transform the C-terminal purification tagged constructs. Our campus is closed from Thursday through the weekend due to the path of Hurricane Lane which delays our lab efforts.

## Highlights

* [Continued efforts to transform C-terminal purification tagged constructs](#subtitle43)
* [Transformation of all N-terminal purification tagged constructs into BL21](#subtitle44)
* [Hurricane Lane delays lab efforts](#subtitle45)

### Continued efforts to transform C-terminal purification tagged constructs

PCR, ligation, and transformation with quantification of DNA on an agarose gel was continued throughout the week. No colonies are observed on any of our plates, indicating that the transformation protocol was conducted incorrectly, ligation was unsuccessful, the PCR produced insufficient amplicons, or the digestion did not work. We previously verified that our RE digest indeed cuts correctly. Ligation and transformation were previously optimized and produced colonies on other constructs. This led us to investigate the PCR itself. The PCR for these constructs is conducted in two steps to add the C-terminal purification tag through the primer.

### Transformation of all N-terminal purification tagged constructs into BL21

All N-terminal tagged constructs were sequence verified from DH5a plasmids, then transformed into BL21. Colony PCR revealed that correct insert sizes were present in a majority of colonies.

### Hurricane Lane delays lab efforts

The onset of Hurricane Lane closed our campus facilities from Thursday through Sunday, pushing our induction and C-terminal transformation plans to the next week.

# AUG 27 - AUG 31

Induction of all N-terminal purification tagged constructs at 28C were carried out for subsequent purification, dialysis and VLP assembly. In addition, PCR amplicons were sent to sequencing to verify that the correct ends and restriction sites were being formed.

## Highlights

* [Induction of N-terminal purification tagged constructs at 28C](#subtitle46)
* [Confirming PCR amplicons of C-terminal tagged constructs](#subtitle47)

### Induction of N-terminal purification tagged constructs at 28C

Induction at 28C for four hours produced proteins of the correct molecular weight. 50 mL cultures were spun down and pellets were stored at -80C until ready for purification.

### Confirming PCR amplicons of C-terminal tagged constructs

Previous efforts to transform C-terminal purification tagged constructs into DH5a continued to be unfruitful. We decided to send the amplicons to sequencing and received poor results.

# SEP 3 - SEP 7

The pellets from our induction of N-terminal purification tagged proteins were purified and stored at -80C in a general dialysis buffer. We determined which buffers we would try for VLP assembly and planned and prepared the materials we needed.

## Highlights

* [N-terminal purification tagged protein purification and planning for VLP assembly](#subtitle48)

### N-terminal purification tagged protein purification and planning for VLP assembly

We purified N-terminal purification tagged proteins and obtained nanodrop readings of 0.66 mg/ml - 1.44 mg/ml for all purified proteins. We will begin to assemble VLPs using these proteins by subjecting them to dialysis buffers of different compositions. Our assays will test for the effects of salt concentration and pH on VLP assembly and if VLP formation is affected by the different CA cut sites or by the addition of a few extra amino acids at the end of our proteins.

# SEP 10 - SEP 14

Our first VLP assembly produces virus-like particles viewable under the EM. We attempt to assemble VLPs with the rest of our N-terminal purification tagged constructs. We had to perform another transformation of BBa\_E1010 into DH5a and received an accurate sequencing result.

## Highlights

* [VLP assembly and our first look at virus-like particles](#subtitle49)
* [Follow up VLP assembly assay](#subtitle50)
* [Cloning and sequencing of BBa\_E1010 into DH5a](#subtitle51)

### VLP assembly and our first look at virus-like particles

VLPs are viewed under the EM by Ryan on our full Gag construct. The particle diameter ranges from ~30 - 50 nm. Cleavage of the His tag appears to form more consistent, spherical, and closed particles. Meanwhile, particles with the His tag still attached seem to have more variability in size with some particles seemingly opened or deflated. Furthermore, we saw more VLPs under acidic assembly buffer conditions than basic conditions, suggesting that pH may play a role in VLP assembly.

### Follow up VLP assembly assay

We decided to follow up on the last VLP experiment by observing the VLPs and intermediates at different points during assembly. We used our full Gag protein for this time assay. We also sought statistical data on VLP sizes to compare which was larger, tagged or untagged Gag. Unfortunately, we could not gather statistical data on VLP sizes as very little VLPs formed from this assembly. Our positive control, a VLP we assembled previously, did not produce as many particles as expected. This suggests that something may have gone wrong in the assembly process.

### Cloning and sequencing of BBa\_E1010 into DH5a

DH5a bacterial colonies that were transformed with BBa\_E1010 during the summer were not preserved, thus we had to perform the transformation again and send the plasmid to sequencing. Sequencing confirmed that we had the correct RFP sequence with the prefix and suffix.

# SEP 17 - SEP 21

Efforts to create the Gag-RFP and Gag biobrick are continued. Sequences were amplified for subsequent digestion and ligation into the biobrick (pSB1C3) and expression (pET14b) vectors.

## Highlights

* [Amplification for expression and Biobrick creation](#subtitle52)

### Amplification for expression and Biobrick creation

The Gag and RFP were amplified off of our Gag construct and BBa\_E1010 sequence in two different ways. For ligation into the pSB1C3 vector for biobrick submittal, the Gag and RFP were amplified with primers consisting of the prefix and suffix. For ligation into the pET14b vector for expression, the Gag and RFP were amplified to incorporate the NcoI and BamHI restriction enzyme sites upstream of the Gag and downstream of the RFP.

# SEP 24 - SEP 28

Amplicons for biobrick and expression were digested, cleaned, quantified, and ligated into their respective vectors.

## Highlights

* [Transformation of DH5a with the Gag insert in pSB1C3](#subtitle53)
* [Transformation of DH5a with the Gag, RFP, and pSB1C3 / pET14b insert](#subtitle54)

### Transformation of DH5a with the Gag insert in pSB1C3

The Gag with prefix and suffix primers was digested with EcoRI and PstI for a melting ice ligation with the digested pSB1C3 vector. Transformation of competent DH5a cells with the Gag insert in the biobrick submittal plasmid appeared successful after colony PCR and sequencing.

### Transformation of DH5a with the Gag, RFP, and pSB1C3 / pET14b insert

The ligation with multiple inserts (Gag and RFP) was something we tried for the first time. We followed the PCR-after-ligation method (An et. al, 2010) and attempted to conduct a PCR on the ligation product with primers flanking our insert (VF2 and VR primers) to obtain the ligated Gag-RFP. The PCR did not show indicative bands of our ~2.1 kb insert. We thus switched gears and attempted to use our Gag ligated to pSB1C3 as the vector backbone.

# OCT 1 - OCT 5

We digested the sequenced Gag in pSB1C3 with SpeI and PstI (RE sites downstream of the Gag) to ligate the RFP digested with XbaI and PstI. This would allow us to obtain the Gag-RFP biobrick and use it as template for amplifying the sequence with the correct RE sites for ligation into pET14b.

## Highlights

* [Digestion and ligation of the RFP into pSB1C3-Gag](#subtitle55)
* [Preparation of biobricks for submittal to iGEM](#subtitle56)

### Digestion and ligation of the RFP into pSB1C3-Gag

The ligation was conducted with a 3:1 insert to vector ratio. Ligation product diluted in TE buffer was used to transform competent DH5a cells. A colony PCR of the Gag-RFP with flanking insert primers (VF2 and VR) revealed a band corresponding to the approximately 2.1 kb insert.

### Preparation of biobricks for submittal to iGEM

We begin to prepare the biobricks for submittal by performing plasmid extractions and following iGEM’s protocol for drying the samples. Unfortunately we did not have time to sequence the Gag-RFP part before submitting.

# OCT 8 - OCT 12

The Gag-RFP in pSB1C3 was used as template for the amplification of the Gag-RFP with primers that would add the NcoI and BamHI RE sites for digestion and ligation into pET14b. However, odd sequencing results of our plasmids after transformation suggest we may have the wrong insert.

## Highlights

* [Amplification of the Gag-RFP for ligation into pET14b](#subtitle57)
* [Odd sequencing results of pET14b containing the Gag-RFP suggest an incorrect insert](#subtitle58)
* [Expression of BL21 cells with large insert reveal no indicative bands on a SDS-PAGE gel](#subtitle59)

### Amplification of the Gag-RFP for ligation into pET14b

The Gag-RFP was amplified with primers to add on the NcoI site upstream of the Gag and the BamHI site downstream of the RFP. The amplicons were gel extracted and digested using the NcoI and BamHI restriction enzymes. Following spin column cleanup, the Gag-RFP was ligated into digested pET14b in a 3:1 insert to vector ratio.

### Odd sequencing results of pET14b containing the Gag-RFP suggest an incorrect insert

Transformation of DH5a and BL21 with the Gag-RFP insert in pET14b appeared to be successful after colony PCR. However, sequencing results of the plasmid with VF2 and VR primers reveal the beginning and end of the Gag. The Gag itself is ~1.3 kb, which does not match up with the insert size observed on the gel.

### Expression of BL21 cells with large insert reveal no indicative bands on a SDS-PAGE gel

BL21 cells with the supposed Gag-RFP insert are expressed with 0.5 mM IPTG for 2 and 4 hours. No band differences are observed between the induced and uninduced cultures.

# Oct 15 - Oct 19

The investigation continues to understand what the insert could possibly be, if not the Gag-RFP fusion protein. Sequencing is conducted with additional primers and steps are redone to try to transform BL21 and DH5a cells with the correct Gag-RFP insert.

## Highlights

* [Sequencing with the VR and RFP reverse primer gives mixed results](#subtitle60)
* [Next steps to produce the Gag-RFP fusion protein](#subtitle61)

### Sequencing with the VR and RFP reverse primer gives mixed results

Another ligation of the pSB1C3-Gag and the RFP was conducted. VF2 and VR primers were used to amplify the insert. This amplicon was sent to sequencing with the VF2, VR, and RFP reverse primer with the BamHI restriction site. The sequencing result from the VF2 revealed the beginning of the Gag as expected. However, the VR and RFP reverse primer gave different results. The RFP reverse primer revealed the end of the RFP as expected, while the VR primer, which also should have revealed the end of the RFP, displayed the end of the Gag. After looking to see if anyone had similar problems on iGEM’s website, we saw documentation that the VR may misprime on certain biobricks. We checked our Gag-RFP sequence against the VR primer and found 5 sites where the VR primer could possibly anneal with 9-12 base pairs complementarity out of its 20 base pairs.

### Next steps to produce the Gag-RFP fusion protein

We continued through with this sequenced VF2 and VR amplicon and proceeded to digest and ligate it into pET14b. Colony PCR again revealed the rather large, ~2.1 kb insert. However, expression in BL21 cells did not display our protein of interest. We plan to backtrack and analyze at which step something could have gone wrong to clone our fusion protein by the time we present at the Giant Jamboree.

While the notebook is temporarily frozen due to the Wiki Freeze, our team will still be busy in the lab! Stay tuned for updates on our final Gag-RFP fusion protein.

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# Team:Hawaii/Experiments

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* ## PROTEASE CLEAVAGE SITE ASSAY

  + I.

    Design of the CR2 Gag-Prot-RT Constructs
  + II.

    CR2 Gag-Prot-RT DH5-a Transformation
  + III.

    CR2 Gag-Prot-RT Induction Optimization
  + IV.

    Tricine SDS PAGE purification of CR2 Gag-Prot-RT 28C and 35C.
  + V.

    CR2 Gag-Prot-RT Purification
  + VI.

    CR2 Gag-Prot-RT Degradation Fragment Extraction for Mass Spectrometry
* ## DESIGNING CONSTRUCTS FOR MATURE VLP FORMATION

  + I.

    Designing CR2 Gag Amplicons
  + II.

    Transformation of Amplicons
* ## VLP FORMATION

  + I.

    Purification of the Various Amplicons Proteins
  + II.

    Assembly of the Purified Proteins using Various Assembly Buffer Conditions
  + III.

    Assembly of the Purified Proteins Constructs and Assembly Time Assays

## INTRODUCTION

Centromere-specific retrotransposons (CRs) are a class of transposable element belonging to the Ty1/Copia and Ty3/Gypsy superfamilies of long terminal repeat (LTR) retrotransposons found predominantly in the centromeres of eukaryotic genomes. CRs are characterized by the chromodomain (CHD) within the C-termini of the integrase; this motif is believed to influence the selectivity of CR insertion at the centromere. Ty3 Gag and Pol genes are arranged in the same order as retroviruses: (1) Gag (2) protease, (3) reverse transcriptase and (4) integrase. On the other hand, Ty1 Gag and Pol genes are ordered: (1) Gag, (2) protease (3) integrase, and (4) reverse transcriptase.

Gag, which forms the structural unit of retroviral and retroviral-like elements, can be further divided into the matrix, capsid, and nucleocapsid domains. The make-up and function of Gag domains varies widely between retroviral and retroviral-like elements. Generally, the matrix facilitates nuclear localization, nucleic acid binding, membrane targeting, and protein-protein interactions [1] the capsid mediates protein-protein interactions leading to Gag multimerization and particle assembly [2], and the nucleocapsid contains a CCHC zinc-finger motif which packages DNA/RNA and facilitates tRNA annealing [3] [4]. Studies investigating the Ty3 Gag polyprotein have shown that Gag is proteolytically processed into a 26-kDa capsid and 9-kDa nucleocapsid [5]. The Gag polyprotein of Ty3/Gypsy retroelements does not contain a matrix domain; however, they do still have the ability to form virus-like particles (VLP). VLPs represent a major intermediate step in retrotransposition reactions [6]. The Saccharomyces Cerevisiae retrotransposon, Ty1, provides a model for elucidating the assembly and structural properties of retro-elements [6]. Genomic Ty elements are first transcribed in the nucleus and then transported into the cytoplasm where the Ty RNA is translated into proteins, the Gag proteins then assemble into VLPs that encapsulate the Ty-RNA, tRNA, and Ty encoded enzymes [6].

Our project is based on the characterization of the CR2-Gag-Pro-RT VLP assembly and packaging capabilities. The first step in this characterization required us to find the specific cut sites of the CR2 protease in order to determine the substrate specificity of these protease enzymes. This determination is critical for giving us insight into the diverse and complex roles the Gag domain has on the formation of VLPs. Using this information we designed CR2 Gag amplicons that translate into proteins which are critical for mature VLP formation. These amplicons were designed with the capsid and nucleocapsid domains in mind. After purifying the proteins created by these amplicons we optimized VLP assembly conditions to determine the most efficient environment for VLP formation. We then assembled and purified VLPs in-vitro using our optimized assembly conditions. The samples were then run through a Native PAGE to determine the approximate VLP size and then visualized using an transmission electron microscope.

### I. Design of the CR2 Gag-Prot-RT Constructs

**Purpose:**  To determine the cleavage sites of the aspartyl-like protease we designed a CR2 Gag-Prot-RT construct which is approximately 2 kb in size.

**Attributions:**  Dan Laspisa gathered the sequences to produce the consensus for the sub-families of CRs in this experiment. Ryan Shontell annotated the consensus sequences, made the predictions, and designed the three constructs with insight from Dr. Gernot Presting.

**Materials and Methods:** Consensus sequences of seven sub-family members of the Zea mays centromere-specific (CR) retrotransposons were assembled by Dan Laspisa.

| Sub-Family Member | Sequences in Consensus |
| --- | --- |
| CR1A | 29 |
| CR1B | 81 |
| CR2 | 171 |
| CR3 | 13 |
| CR4B | 48 |
| CR4R1 | 37 |
| CR5 | 157 |

The consensus sequences were run through the Conserved Domain Basic Local Alignment Search Tool (CD BLAST) to identify conserved domains within the open reading frame of the Gag and Pol polyproteins. The consensus sequences were further analyzed using publicly accessible protein modification servers to identify conserved modifications of the polyproteins. Covalent protein modifications were scanned for using ProtParam (Gasteiger et al., 2005), MyHits Motif Scan (SIB, Switzerland), and PlantsP, (Podell and Gibskov, 2004). Nuclear localization signals were scanned for using NLStradamus (Nguyen et al., 2009), NucPred (Stockholm Bioinformatics Center, Sweden), cNLS Mapper (Kosugi et al., 2009), WoLF PSORT
(CBRC, Japan), and SeqNLS (Lin et al., 2012). These conserved domains were annotated on the consensus sequences using Geneious (Biomatters, Newark, NJ 07102, USA) and an alignment was generated using MUSCLE (EMBL-EBI, England).

Making inferences from previous studies investigating the cleavage specificity of the aspartyl-like proteases of retroviruses, we predicted putative cleavage sites along the CR polyproteins. These studies found that in most cases, aspartyl-like proteases cut substrates at the bond between two adjacent hydrophobic amino acids (Toszer, 2010; Laco, 2015). We scanned through the consensus sequences and noted sites meeting the criteria for adjacent hydrophobic residues. We additionally considered the covalent modifications that have been reported in specific domains. We identified three putative cleavage sits between the Gag and Protease domains, eight between the Protease and Reverse Transcriptase domains, and four between the RNAseH and Integrase domains.

After predicting these domains, the putative mature proteins were modelled separately using I- TASSER (Zhang, 2008), PHYRE2 (Kelley et al., 2015), and HHPred (Max Planck Institute, Germany). The accuracy of the models produced were assessed using ANOLEA (Melo et al., 1997) RAMPAGE (University of Cambridge, England), and Verify3D (Luthy et al., 1992). ANOLEA performs pairwise comparisons of the energy between the heavy atoms of each amino acid in a non-local environment, RAMPAGE performs Ramachandran analysis for the stereochemistry of the amino acid side chains around peptide bonds, and Verify3D compares the 3D model against it’s 1D amino acid sequence and scores the likelihood of a residue being in its structural class.

The top five models from the putative mature proteins were used to make consensus models using MODELLER (Sali Lab, San Francisco, California). Five consensus models were produced for each predicted mature protein. These were checked with ANOLEA, RAMPAGE, and Verify3D and the top scoring models were used for further analysis. X-Ray crystallography models from the Protein Data Bank (Berman et al., 2000) of the Ty3/Gypsy or retroviral equivalents to our predicted mature proteins were downloaded and then compared to our consensus models using PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).

**Results and Discussion:** The comparisons between our predicted 3D models and the X-Ray crystallography data allowed us to further restrict the number of predicted putative cleavage sites based on the locations of terminal alpha helices and their associated covalent protein modifications. The CR2 Gag-Prot-RT construct encompasses the region from Gag to the 5’ tip of the reverse transcriptase domain. The construct was designed with a N-terminal and C-terminal 8-histidine tag to allow for the purification of the regions 5’ and 3’ of the cut site using nickel resin. This constructs was cloned into the pET-14b vector using a 5’ NcoI restriction site and a 3’ BamHI restriction site (GenScript).

| CR2 Gag-Prot-RT | |
| --- | --- |
| Amplicon Size (bp) | 2342 bp |
| Protein Size (kDa) | 90 kDa |

### II. CR2 Gag-Prot-RT DH5-a Transformation

**Purpose:** To ligate the CR2 Gag-Prot-RT into pET-14b vector which will then be used to transform into DH5-α for future experimentation and long-term storage of the plasmid.

**Attributions:** The initial colony PCRs for verification of inserts were performed by Ryan Shontell. Troubleshooting and colony PCR for verification of CR2 Gag-Prot-Rt was performed by Gina Watanabe and Emily Yang. Initial sequencing using the T7 forward and reverse primers were performed by Ryan Shontell. Later sequencing performed using T7 and internal primers ordered from IDT were performed by Gina Watanabe. Analysis of all sequences was performed by John Banasihan, Ryan Shontell, Jonathan Tello, and Dr. Gernot Presting.

**Materials and Methods:** Our transformation protocol began with a double-digestion of a pET-14b vector using BamH1 and NcoI NEB-HF restriction enzymes in preparation for ligation. We then proceeded to ligate our CR2 Gag-Prot-RT insert into the now digested vector. We then used DH5-α competent cells, a pUC19 vector as our control and the CR2 Gag-Prot-RT ligated vector (2-fold TE dilution) to transform into the DH5-α competent cells. The DH5-α cells that grew on the antibiotic agar were then used for a colony PCR.

BLASTN was used to determine whether internal priming of would occur between the CR constructs and the T7 Forward and T7 Reverse primers. It was determined that no significant lengths of the primers would be able to anneal; however, there was the potential for extension of smaller PCR products due to partial annealing.

After running our colony PCR products on an agarose gel we saw that the CR2 Gag-Prot-RT bands were not very strong. We later optimized the PCR parameters by decreasing the annealing temperature from 65°C to 55°C and increasing the extension time. These changes are seen in Figure 3.

After this optimization of the PCR parameters the CR2 Gag-Prot-RT from figures 1, 2, and 3 were sent in for sequencing at the University of Hawaii at Manoa sequencing facility. Glycerol stocks were also prepared from these colonies.

**Results and Discussion:** The decrease in annealing temperature seems to have greatly improved the CR2 Gag-Prot-RT colony PCR; however, much of the smaller bands still appear in the gel all averaging roughly 0.5 – 1 kb. These bands did not appear in the previous gels using high annealing temperatures suggesting that these may be products of internal priming. Even though there are putative internal priming products present, we lose some bands that were present in previous high annealing temperature PCRs. Contamination of the PCR reagents was ruled out as the colony PCR was performed in parallel with the other constructs using the same master mix. The product produced during the colony PCR were then sequence verified before further experimentation.

The CR2 Gag-Prot-RT gels from figures 1-3 showed that the plasmid was indeed taken up by all colonies sent in. As the sequencing had been performed with T7 forward and T7 reverse primers, only the first 500-600 base pairs of the 5’ and 3’ end of the constructs could be verified. We ordered three internal primers for the CR2 Gag-Prot-RT construct to close the gaps for verification of the sequence.

![](https://static.igem.org/mediawiki/2018/2/2f/T--Hawaii--exp_fig1.jpg)

**Figure 1.** Colony PCR of CR2 Gag-Prot-RT with T7 forward and reverse primers. PCR conditions: (1) Initial Denaturation at 94℃ for 30 seconds (2) Denaturation at 94℃ for 20 seconds (3) Annealing at 65℃ for 20 seconds (4) Extension at 68℃ for 1 minute 30 seconds (5) Final Extension at 68℃ for 5 minutes; run for 35 cycles. Amplicon run on 1.5% agarose gel in TAE at 95V. Weak bands of the appropriate size can be seen in colonies 1, 3, and 4. These were used for sequencing.

![](https://static.igem.org/mediawiki/2018/4/49/T--Hawaii--exp_fig2.jpg)

**Figure 2.** Colony PCR of CR2 Gag-Prot-RT with T7 forward and reverse primers with longer extension times. PCR conditions: (1) Initial Denaturation at 94℃ for 30 seconds (2) Denaturation at 94℃ for 20 seconds (3) Annealing at 65℃ for 20 seconds (4) Extension at 68℃ for 2 minute 20 seconds (5) Final Extension at 68℃ for 5 minutes; run for 35 cycles. Amplicon run on 1.5% agarose gel in TAE at 95V, for sequencing. Longer extension times did not improve the PCR significantly.

![](https://static.igem.org/mediawiki/2018/2/27/T--Hawaii--exp_fig3.jpg)

**Figure 3.** Colony PCR of CR2 Gag-Prot-RT with T7 forward and reverse primers with longer extension time and decreased annealing temperature. PCR conditions: (1) Initial Denaturation at 94℃ for 30 seconds (2) Denaturation at 94℃ for 20 seconds (3) Annealing at 55℃ for 20 seconds (4) Extension at 68℃ for 2 minutes 20 seconds (5) Final Extension at 68℃ for 5 minutes; run for 35 cycles. Amplicon run on 1.5% agarose gel in TAE at 95V. Lanes 1, 2, 3, 4, and 7 contain the full CR2 Gag-Prot-RT construct.

### III. CR2 Gag-Prot-RT Induction Optimization

**Purpose:** To optimize the induction conditions for the CR2 Gag-Prot-RT construct. These conditions included time, temperature and IPTG concentration.

**Attributions:** The SDS PAGE runs, inductions, and all subsequent analysis were performed by Ryan Shontell and Gina Watanabe.

**Materials and Methods:** An overnight culture (ON) of the transformed CR2 Gag-Prot-RT BL21 cells was prepared (10 mL of this ON culture will be used to perform a 200 mL induction). 50 μg/μL of chloramphenicol and 50 μg/μL of ampicillin was added to the ON culture. The flask was then left in the incubation shaker ON at 37°C at 220 rpm.

200 mL of media was then added to the induction container along with the 10 mL of the ON culture. The induction culture was then allowed to grow at 37°C. (Note: For 1-15 mL induction cultures it is recommended to allow growth for 1 hour at 220 rpm. For inductions that are 100+ mL it is recommended to allow to growth for 2 hours at 175 rpm.)

Since the induction culture does not receive antibiotics, aseptic techniques are crucial for the following steps. 100 μL of each culture was pipetted into a clean 1.5 mL microcentrifuge tube and spun at 400 rpm for 5 minutes. The supernatant was discarded and the pellet was resuspended in 20 μL of 1X SDS-PAGE Loading Buffer + 5% B-ME. The tubes were then heated at 95°C for 5 minutes to help with the resuspension. To the induction culture, between 0.2-1 mM of IPTG was added and incubated at the optimized temperature for 2-10 hours. To determine the optimized conditions for induction, 100 μL of culture was collected every hour and then analyzed using an SDS-PAGE.

**Results and Discussion:**  In this experiment we optimized the conditions for the induction of the CR2 Gag-Prot-RT Construct. The full size CR2 Gag-Prot-RT will appear on a gel at ~90 kDa. Putative degradation products occur at ~75 kDa and ~50 kDa. We predict that these bands correspond with the full-size Gag (~52-54 kDa) and Gag-Protease (54-75 kDa). Since the protease appears to be active at these temperatures, we will perform additional inductions at other temperatures (20°C - 28°C). To see if the number or intensity of putative degradation products changes. Under these induction parameters at 35°C a significant quantity of the full-sized construct is expressed. We then used these parameters to induce and purify this CR2 Gag-Prot-RT fragment.

![](https://static.igem.org/mediawiki/2018/3/36/T--Hawaii--exp_fig4.jpg)

**Figure 1.** CR2 Gag-Prot-RT Induction at 32℃ and 35℃. Induction performed with 05 mM IPTG for 6 hours at a temperature of 32℃ or 35℃. (1) Marker [Precision Plus Protein Dual COlor Standard] (2) 32℃ Uninduced at 0 hours (3) 32℃ Induced at 6 hours (4) 32℃ Induced at 2 hours (5) 32℃ Induced at 6 hours (6) 35℃ Uninduced at 0 hours (7) 35℃ Uninduced at 6 hours (8) 35℃ Induced for 2 horus (9) 35℃ Induced for 6 hours. Full size CR2 Gag-Prot-RT occurs at ~90 kDa. Putative degradation products occur at 75 kDa and 50 kDa. We predict that these bands correspond with the full-size Gag (~52-54 kDa) and the Gag-Protease (~54-75 kDa).

### IV. Tricine SDS PAGE purification of CR2 Gag-Prot-RT 28℃ and 35℃

**Purpose:** For the CR2-Gag-Pro-RT proteins induced at 28℃ and at 35℃, a Tricine Gel run based on Haider et al., 2012’s protocols was performed. The tricine gel was used to separate proteins of 5-30 kDa or less. This will separate the RT protein from the full Gag protein and allow us to extract gag via gel extraction.

**Attributions:**  All experiments conducted relating to the Tricine gel purification was done by John Banasihan.

**Materials and Methods:**  In this experiment Haider et al., 2012’s protocols were followed with some minor adjustments due to availability of resources. 10% APS was used to make both the stacking and resolving gels. The stacking gel consisted of 0.76 ml of 2.5M Tris (pH 8.8), 3.42 ml DI H 2 O, 150 μl APS, 0.66 ml of acrylamide bisphosphate (29:1, 30%), and 5.0 μl of TEMED. The Resolving gel consisted of 5.6 ml of 2.5 M Tris (pH 8.8), 0.90 ml of DI H 2 O, 3.33 ml of AB (29:1, 30%), 150 μl of APS, 6.0. A running buffer was made of 3.03 g of Tris base, 4.5 g Tricine, 0.5 g SDS in 1 L of DI water.

**Results and Discussion:**  The Tricine gel does not show a clear separation of proteins 5-30 kD in size. While there are some bands that are indicative of the presence of Gag and the Gag-Pro polyprotein, there appears to be no signs of the RT protein. The Tricine gel experiment was unsuccessful in separating the protease and the smaller RT protein. This may have been due to the modifications made to Haider et al., 2012’s protocols, such as running the gel at 70V stacking and 150V running rather than the continuous 125-150V, however, these modifications should not have significantly impacted the overall experiment. The protease cleavage sites will be determined via mass spectrometry and trypsin digest rather than redoing another tricine gel experiment, since the mass spectrometry will offer more accurate results.

![](https://static.igem.org/mediawiki/2018/4/44/T--Hawaii--exp_fig5.jpg)

**Figure 1.**  CR2-Gag-Prot-RT 35 C; Inclusion body purification without lysozyme run on a tricine gel, (4% Stacking, 10% resolving). The gel does not appear to be able to successfully separate proteins of 5-30 kDa or less as the lowest readable bands are around 50 kDa, indicative of the Gag protein.

![](https://static.igem.org/mediawiki/2018/d/dd/T--Hawaii--exp_fig6.jpg)

**Figure 2.**  CR2-Gag-Prot-RT 28C; inclusion body purification without lysozyme run on a tricine gel,
(4% Stacking, 10% resolving). The gel does not appear to be able to successfully separate
proteins of 5-30 kDa or less as the lowest readable bands are around 50 kDa, indicative of the
Gag protein.

### V. CR2 Gag-Prot-RT Purification

**Purpose:** To produce higher quality SDS-PAGE gel images of the soluble vs. insoluble fractions and to determine if enough expressed protein may be purified from the soluble fraction for future work.

**Attributions:**  Purifications were performed by Ryan Shontell, Gina Watanabe, Jonathan Tello and John Banasihan.

**Materials and Methods:**  A soluble protein purification on the CR2 Gag-Prot-RT was performed according to our standard protocol. The proteins collected for this experiment were induced at 28°C and 35°C. This was not done under denaturing conditions as there was no urea present in the in buffers used. Both the soluble and insoluble proteins were collected and tested on an SDS PAGE.

**Results and Discussion:**  Gels comparing the soluble and insoluble fractions of CR2 Gag-Prot-RT were run to generate higher quality gels to determine which fraction the expressed proteins occur and to determine whether smaller degradation fragments of the CR2 Gag-Prot-RT construct become soluble.

Figure 1 depicts the soluble vs. insoluble fractions of CR2 Gag-Prot-RT induced at 28°C and 35°C. The putative 90 kDa full sized CR2 Gag-Prot-RT construct, 75 kDa CR2 Gag-Prot degradation fragment, and 50 kDa CR2 Gag degradation fragment occur in the insoluble fraction. Another interesting finding to note is the difference in intensity of the 75 and 50 kDa bands between the 28°C and 35°C inductions. It is possible that the protease is more active at lower temperatures and is there for able to cleave the 75 kDa intermediate into the 50 kDa fragment at a higher rate leading to the loss of the 75 kDa band and gain of a more intense 50 kDa band at 28°C. These findings suggest that a lower temperature induction should be conducted to screen for the smaller fragments of the construct corresponding to protease, RT tail, capsid, spacer, and nucleocapsid. In future insoluble purifications to screen for these smaller fragments, lysozyme should be avoided as it generates a very intense band in the 10-15 kDa region. We believe that the RT tail, protease, and nucleocapsid proteins are roughly this size. Protease, nucleocapsid, and spacer by themselves are not histidine tagged and so, would likely be found in the washes of a purification whereas the capsid and RT tail would be found in the elutions.

Figure 2 depicts the purification performed on the soluble fraction of the CR2 Gag-Prot-RT 28°C and 35°C cultures. Small amounts of the full-sized fragment do occur in the soluble fraction; however, the yield is very low and it is inadvisable to attempt future purifications form the soluble fraction. Washes were performed with wash buffer containing no imidazole. Future purifications will utilize 10mM and 30mM wash buffers to optimize the removal of non-histidine tagged proteins form the nickel resin.

![](https://static.igem.org/mediawiki/2018/d/d2/T--Hawaii--exp_fig7.jpg)

**Figure 1.** CR2 Gag-Prot-RT 28℃ / 35℃ vs. Insoluble fractions. (1) Marker [Plus Protein Dual COlor Standard] (2) 28℃ Total Protein (3) 28℃ Soluble (4) 28℃ Insoluble (5) Empty (6) 35℃ Total Protein (7) 35℃ Insoluble. 5% stacking gel at 80V; 15% resolving gel 100V. The majority of induced protein occurs in the insoluble fraction. Note that possible degradation products at 75 and 50 kDa.

![](https://static.igem.org/mediawiki/2018/5/54/T--Hawaii--exp_fig8.jpg)

**Figure 2.** CR2 Gag-Prot-RT 28℃ / 35℃ Soluble Protein Purification. (1) Marker [Plus Protein Dual Color Standard] (2) 28℃ Total Protein (3) 28℃ Wash (4) 28℃ Elution (5) 28℃ Elution 2 (6) 35℃ Wash 1 (7) 35℃ Elution 1. (8) 35℃ Elution. 1.5% stacking gel at 70V; 15% resolving gel at 100V. 5% stacking gel at 80V; 15% resolving gel at 100V. Washed were performed with wash buffers without imidazole. 250 mM imidazole elution buffer was used.

### VI. CR2 Gag-Prot-RT Degradation Fragment Extraction for Mass Spectrometry

**Purpose:**  To perform gel extraction of the putative degradation fragments of CR2 Gag-Prot-RT to send to Taplin for analysis via mass spectrometry in order to determine the cleavage sites of CR2 Gag-Prot-RT construct.

**Attributions:**  Gel extraction was performed by Vishal Negi and Ryan Shontell.

**Materials and Methods:**  A glass plate was sterilized with 70% ethanol and separate surgical blades were prepared for each band we excised from the gels. The desired SDS-PAGE gel was then placed on the glass plate and the band of interest was excised. The excised band was then placed into a 1.5 mL microcentrifuge tube and 1 mL of miliq H2O was added. The samples were then labeled and the caps were wrapped in parafilm. Bands were extracted from three separate gels. Gels were selected by Vishal Negi based on the resolution of the gel and intensity of the bands. The 90 kDa band likely corresponds to the full-sized CR2 Gag-Prot-RT. We believe that Band 2 (75 kDa) and Band 3 (50 kDa) are degradation products of the full-sized construct corresponding to CR2 Gag-Prot and CR2 Gag, respectively. Data from the mass spec will provide us with the cleavage sites necessary to accurately produce future constructs of the mature Gag and mature protease.

**Results and Discussion:**  To determine protease cleavage sites, the ~100 kDa, ~75 kDa, and ~50 kDa bands representing the full polyprotein (CR2 Gag-Protease-RT ~90 kDa), the partial polyprotein (CR2 Gag-Protease ~62-77 kDa), and the full gag (~52-54 kDa) respectively were extracted from three separate gels of cells induced at 35℃ selected by Vishal Negi and Ryan Shontell. Selection was based off of resolution of the gel and band intensity (Fig. 1). Samples were sent to the Taplin Mass Spectrometry Facility on June 1, 2018, and received by the facility on June 5, 2018. Results were obtained on June 29, 2018. We were notified that the samples were contaminated and contained fragments from all three bands. However, filtering allowed for the determination of the most relevant fragments for each sample.

Tryptic fragments for each band were mapped on to the CR2 Gag-Protease-RT GenScript construct using fragment data copied from the Taplin results page into an Excel sheet and sorted by position. The 100 kDa fragments covered 90.04 kDa, the 75 kDa fragments covered 56.76 kDa, and the 50 kDa fragments covered 47.88 kDa of the full construct. These fragments confirmed a protease cleavage site downstream of the nucleocapsid in the form of an 18 bp gap between the end of the last 50 kDa fragment (6 bp upstream of the 3’ end of the CCHC Zinc Finger domain) and the beginning of the next 75 kDa and 100 kDa fragments (12bp after the 3’ end of the CCHC Zinc Finger). The second cleavage site between the protease and reverse transcriptase domains could not be identified as the 75 kDa fragments did not exceed 56.76 kDa, with the most upstream protease cut site observed being located well within the protease domain. This suggests that either the wrong band was excised from the PAGE gel or that the protease has auto digestive activity under the conditions used.

![](https://static.igem.org/mediawiki/2018/5/54/T--Hawaii--exp_fig9.jpg)

**Figure 1.** Image of gel and corresponding bands sent to Taplin for analysis by mass spectrometry. Bands were excised from the gel and then placed into 1.5 mL microcentrifuge tubes with 1 mL of Miliq H2O. Samples were shipped to Harvard on 6-1-18 and received at the facility 6-5-18.

![](https://static.igem.org/mediawiki/2018/2/2a/T--Hawaii--exp_fig10.jpg)

**Figure 2.** Image of gel and corresponding bands saved for analysis by mass spectrometry at the local proteomics facility. Bands were excised from the gel and then placed into 1.5 mL microcentrifuge tubes with 1 mL of Miliq H2O. Samples were shipped to Harvard on 6-1-18 and received at the facility 6-5-18.

# DESIGNING CONSTRUCTS FOR MATURE VLP FORMATION

### I. Designing CR2 Gag Amplicons

**Purpose:**  To design amplicons that will create the proteins necessary for VLP formation. Within the Gag region of this CR2 construct lies a capsid region (CA) and nucleocapsid region (NC). As we are unsure of the role of the CA and NC regions in VLP formation we created amplicons depending on where the protease cut sites are.

**Attributions:**  These amplicons were designed by Gina Watanabe and finalized by the entire team, including Dr. Gernot Presting and Ryan Shontell.

Materials and Methods: To design amplicons that will create proteins necessary for VLP formation we began by using the original CR2 Gag-Prot-RT sequence. Figure 1 portrays the relationship between the Gag domain and the protease along with the N-terminus and C-terminus TEV/His-tag regions. We amplified off of this sequence to obtain the amplicons described in Table 1.

![](https://2018.igem.org/File:T--Hawaii--exp_fig11.jpg)

**Figure 1.** Picture depicting the CR2 construct with the protease. Also highlights the the N-terminus and C-terminus TEV and HIS Tag regions.

**Table 1.**  Table of all of the different amplicons that were created. The NcoI and BamH1 are the restriction enzyme cut sites used for the ligation protocol. HisTEV is a tag used as a key element in the protein purification i.e. the nickel column we use binds to the His region and the TEV is another restriction enzyme site used to cut off the His region when it is no longer needed. CA stands for capsid; NC stands for nucleocapsid; CA 1, 2, and 3 are different portions of the construct obtained by looking at different subfamilies to determine a consensus. The blue highlighted portion represents the N-terminal amplicons and the red represent the C-terminal.

| Assigned # | Amplicon | Size (bp) |
| --- | --- | --- |
| 1 | NcoI + HisTEV - CA - NC + BamH1 | 1304 |
| 1a | NcoI + HisTEV - CA - NC + Stop + BamH1 | 1307 |
| 2 | NcoI + HisTEV - CA1 + BamH1 | 971 |
| 2a | NcoI + HisTEV - CA1 + Stop + BamH1 | 974 |
| 3 | NcoI + HisTEV - CA2 + BamH1 | 1154 |
| 3a | NcoI + HisTEV - CA2 + Stop + BamH1 | 1157 |
| 3.5a | NcoI + HisTEV - CA3 + Stop + BamH1 | 1187 |
| 4a | NcoI + CA - NC + TEVHis + Stop + BamH1 | 1298 |
| 5a | NcoI + CA1 + TEVHis + Stop + BamH1 | 965 |
| 7a | NcoI + CA3 + TEVHis + Stop + BamH1 | 1178 |

**Results and Discussion:**  To be able to successfully amplify the C-terminus amplicons, a two step PCR was performed to sequentially add a C-terminal purification tag consisting of a TEV cleavage site and an 8X histidine-tag.

The CA-NC amplicon the cut site was determined using the mass spectrometry results from the protease cut site experiments (shown in Fig 2). For amplicons CA1, CA2, and CA3 the 3’ cut sites were determined by looking at different subfamilies of centromeric retrotransposons to determine a consensus at which 3 hypotheses were then determined. Fig 2 is the visualization of Fig 1, showing the relationship between the 4 different amplicons.

![](https://2018.igem.org/File:T--Hawaii--exp_fig12.jpg)
![](https://static.igem.org/mediawiki/2018/5/56/T--Hawaii--exp_fig13.jpg)

**Figure 2.**  Figures showing the relationship between the CA-NC, CA1, CA2, and CA3 amplicons. These figures depict the different cut sites on the 3’ end of the construct done by the protease, determining where the amplicon will begin and end. Base 125-600 were cut out to fit on page.

### II. Transformation of Amplicons

**Purpose:**  Once successfully transformed into BL21 we will express the proteins and purify them for VLP formation experimentation.

**Attributions:**  Protocol for the N-terminus amplicon transformation were done by John Banasihan, and the C-terminus by Gina Watanabe. A second attempt for the C-terminus was conducted by Jonathan Tello and Shelby Roberson.

**Methods and Materials:**  Constructs 1-3.5a were amplified off the CR2 Gag-Pro-RT sequence and then digested with NcoI and BamHI restriction enzymes for ligation into pet14b according to our standard protocol. The ligated products were used to transform a DH5a strain and a colony PCR determined which colonies were had successfully uptaken the correct amplicon. The successful colonies were then selected for plasmid preparation in order to transform BL21 strains. A colony PCR would confirm the colonies that had been successfully transformed.The respective protein was purified from the successful colonies and then prepared for VLP transformation.

**Results and Discussion:**  We were able to successfully express constructs 1, 2, 2a, 3a, and 3.5a and we purified them in protein form. The C-terminal construct transformations were unsuccessful. In regards to protocols used for the N-terminus and the C-terminus constructs, we used a 2-step PCR to generate C-terminus amplicons. This was the only difference yet transformation into the DH5a for the C-terminus was unsuccessful. C-terminal construct transformation into DH5a was attempted multiple times and every attempt was unsuccessful. Some potential reasons for this are: PCR cycles were not optimal, unsuccessful second step PCR, contamination that interfered with the PCR reaction, DH5a cells were no longer competent, and the restriction enzymes did not properly digest the 2nd step PCR amplicons as the digested products did not properly appear on the agarose gel as expected.

The following figures are the agarose gel runs of the colony PCR for transformed BL21 strains with different constructs. Figure 1 shows the results for the BL21 colony PCR of constructs of constructs 1 and 2a. Construct 1 appeared on the gel while construct 2a did not. Construct 2a BL21 transformation was repeated until successful colonies were determined (Figure 3). Figure 2 shows the results for the BL21 colony PCR of constructs 1, 3.5a, and 7. Construct 1 appears on the gel while amplicons 3.5a and 7 did not. Possible reasons for this are mentioned in the above paragraph. Since neither 3.5a and 7 did not show up, another transformation process was done. Figure 3 shows the results for the BL21 colony PCR of constructs of constructs 3a and 2a. Both constructs show up in almost all lanes indicating that all of the colonies that grew contained the inserted gene. These colonies were then induced to created their 3a and 2a proteins and were purified and stored away for further VLP experimentation. Figure 4 shows the results for the BL21 colony PCR of constructs of 3a and 3.5a. Both constructs show up in almost all lanes indicating that all of the colonies that grew contained the inserted gene. These colonies were then induced to created their 3a and 3.5a proteins and were purified and stored away for further VLP experimentation.

![](https://static.igem.org/mediawiki/2018/0/03/T--Hawaii--exp_fig14.jpg)

**Figure 1.**  An agarose gel of the PCR products from BL21. The top portion of the gel is are the products of a colony PCR of BL21 transformed with construct 1. The bottom portion of the gel are the products of a colony PCR of BL21 transformed with construct 2a. The gel was run on a 1% agarose gel at 90V for 40 minutes.

![](https://static.igem.org/mediawiki/2018/8/83/T--Hawaii--exp_fig15.jpg)

**Figure 2.**  An agarose gel of the PCR products from BL21. The top portion of the gel is are the products of a colony PCR of BL21 transformed with construct 1. The bottom portion of the gel are the products of a colony PCR of BL21 transformed with construct 7 in lanes 2-6 and construct 3.5a in lanes 7-8 . The gel was run on a 1% agarose gel at 90V for 40 minutes.

![](https://2018.igem.org/File:T--Hawaii--exp_fig16.jpg)

**Figure 3.**  An agarose gel of the PCR products from BL21. The top portion of the gel is are the products of a colony PCR of BL21 transformed with construct 3a. The bottom portion of the gel are the products of a colony PCR of BL21 transformed with Construct 2a. The gel was run on a 1% agarose gel at 90V for 40 minutes.

![](https://static.igem.org/mediawiki/2018/b/b1/T--Hawaii--exp_fig17.jpg)

**Figure 4.**  An agarose gel of the PCR products from BL21. The top portion of the gel is are the products of a colony PCR of BL21 transformed with construct 3a. The bottom portion of the gel are the products of a colony PCR of BL21 transformed with construct 3.5a . The gel was run on a 1% agarose gel at 90V for 40 minutes.

# VLP FORMATION

### I. Purification of the Various Amplicons Proteins

**Purpose:** To purify all various gag protein constructs and prepare them for VLP assembly. We will store these proteins in the designated VLP assembly buffer in -80 C.

**Attributions:** John Banasihan was the primary lead on the VLP formation experiments. Ryan Shontell assisted him in the imaging of the VLPs.

**Materials and Methods:**  ON cultures of each protein construct were prepared and each was induced in 100 ml cultures for 2 hours at 28oC. Insoluble protein purification was performed under denaturing conditions. Each proteins was washed with denaturing wash buffer and denaturing elution buffer three times. Each wash and subsequent elution were ran on a 15% PAGE gel to confirm the presence of the target protein.

**Results and Discussion:**  The following images are SDS PAGE runs of the purification process. The now purified proteins were used for further experimentation. All of the previous amplicons stated above were successfully purified. Each construct had bands corresponding to the correct protein size. The following figures represent the constructs 1, 2a, 3, 3a, and 3.5a purified proteins.

![](https://static.igem.org/mediawiki/2018/9/92/T--Hawaii--exp_fig18.jpg)

**Figure 1.**  Construct 1 purified from BL21 culture induced at 28℃. Expected size of 48.90 kDa matches with the observed bands of protein on the gel. This was run at 70V stacking for 20 minutes and 100V resolving for 60 minutes on a 15% PAGE.

![](https://static.igem.org/mediawiki/2018/6/6c/T--Hawaii--exp_fig19.jpg)

**Figure 2.**  Construct 2a purified from BL21 culture induced at 28℃. Expected size of 36.98 kDa matches with the observed bands of protein on the gel. This was run at 70V stacking for 20 minutes and 100V resolving for 60 minutes on a 15% PAGE.

![](https://static.igem.org/mediawiki/2018/9/98/T--Hawaii--exp_fig20.jpg)

**Figure 3.**  Construct 3 purified from BL21 culture induced at 28℃. Expected size of 43.44 kDa matches with the observed bands of protein on the gel. This was run at 70V stacking for 20 minutes and 100V resolving for 60 minutes on a 15% PAGE.

![](https://static.igem.org/mediawiki/2018/2/2f/T--Hawaii--exp_fig21.jpg)

**Figure 4.** Construct 3a purified from BL21 culture induced at 28℃. Expected size of 43.44 kDa matches with the observed bands of protein on the gel. This was run at 70V stacking for 20 minutes and 100V resolving for 60 minutes on a 15% PAGE.

![](https://static.igem.org/mediawiki/2018/8/88/T--Hawaii--exp_fig22.jpg)

**Figure 5.** Construct 3.5a purified from BL21 culture induced at 28℃. Expected size of 43.44 kDa matches with the observed bands of protein on the gel. This was run at 70V stacking for 20 minutes and 100V resolving for 60 minutes on a 15% PAGE.

### II. Assembly of the Purified Proteins using Various Assembly Buffer Conditions

**Purpose:** To optimize gag VLP assembly and determine if assembly is favorable under acidic or basic conditions and to determine the effect of having a 8X His-tag on VLP Assembly.

**Attributions:** John Banasihan followed a modified version of Ryan’s protocol on VLP assembly and created an assay that tested the tagged and untagged Capsid only protein (Construct 3a).

**Materials and Methods:**  A 200 ml induction was performed for construct 3a (HisTEV-CA2). The inclusion bodies were purified according to standard protocol without lysozyme. The proteins were separated for further His-cleavage. For the tagged proteins, a buffer exchange was immediately performed to suspend the proteins in the three different VLP buffers, acidic, neutral, and basic. The proteins selected for His cleavage were dialyzed with non-denaturing wash buffer and then cleaved with AcTEV protease overnight. The cleaved proteins were then dialyzed with the three different VLP buffers. All protein concentration were determined via nanodrop and 100 ng from each protein would be used for VLP assembly.

VLP Assembly reactions would contain 100 ng of each protein, 20 mM DTT, 200 mM MgCl2, and the protein’s VLP assembly buffer to fill up to 20 μl. Once the DTT and MgCl2 were added, the assembly reactions were incubated in a water bath at 30 C for 3 hours. Once complete, 2 μl of 100% glycerol were added. 6 μl of each sample were taken for observation under the EM at the University of Hawaii and Manoa.

**Results and Discussion:** The VLP assay tested for the effectiveness of VLP assembly under these conditions: (1) The effect of acidity and (2) the effect the presence of a His-tag has on VLP assembly. First, we were able to confirm that our gag protein variant, construct 3a (CA2) could form VLP’s. The EM images of the CA2 VLPs revealed larger VLP formation under acidic conditions and little to no formation under basic conditions (Figure 1 and 3). Under basic conditions, the EM images showed mainly VLP intermediates of the capsid proteins. This suggests that the gag VLP formation may be hindered but not completely prohibited under basic conditions. Both tagged and untagged VLPs formed under acidic conditions and neutral conditions (Figure 1 and 2). This suggests that tagged capsid proteins do not entirely prohibit VLP assembly. The average diameter size for the tagged capsid proteins assembled in acidic conditions was 42.31 nm while the average diameter size for the untagged capsid proteins was 36.11 nm (Table 1). However, the sample size was too small to perform a true statistical analysis, so these are mainly observations.

**Table 1.** Average VLP diameter for all samples. Each sample size (n) is different. Acidic-tagged n = 10, acidic-untagged n = 8, neutral untagged n = 3, and basic-tagged n = 5.

| Assembly Buffer pH | Tagged VLP diameter (nm) | Untagged VLP diameter (nm) |
| --- | --- | --- |
| acidic | 42.31 | 36.11 |
| neutral | No VLPs | 28.8 |
| basic | 32.96 | No VLPs |

![](https://static.igem.org/mediawiki/2018/4/40/T--Hawaii--exp_fig23.jpg)

**Figure 1** Construct 3a with no His-tag using an assembly buffer with acidic pH. VLPs were imaged in the same buffer they were assembled with. This image was captured on a scale of 50 nm at 40,000x magnification using the Biological Electron Microscopy Facility at the University of Hawaii at Mānoa.The VLPs measured are 33.4 nm, 37.1 nm, 32.2 nm, 33.2 nm, 39.5 nm.

![](https://static.igem.org/mediawiki/2018/4/42/T--Hawaii--exp_fig24.jpg)

**Figure 2.** Construct 3a with His-tag using an assembly buffer with acidic pH. VLPs were imaged in the same buffer they were assembled with. This image was captured on a scale of 50 nm at 50,000x magnification using the Biological Electron Microscopy Facility at the University of Hawaii at Mānoa.The VLP’s measured out in the image have diameters of 28.1 nm, 44.7 nm, 44.9 nm, and 40.8 nm.

![](https://static.igem.org/mediawiki/2018/d/df/T--Hawaii--exp_fig25.jpg)

**Figure 3.** Construct 3a with a His-tag using an assembly buffer with basic pH. VLPs were imaged in the same buffer they were assembled with. This image was captured on a scale of 50 nm at 50,000x magnification using the Biological Electron Microscopy Facility at the University of Hawaii at Mānoa.

![](https://static.igem.org/mediawiki/2018/e/e2/T--Hawaii--exp_fig26.jpg)

**Figure 4.** Construct 3a with no His-tag using an assembly buffer with basic pH. VLPs were imaged in the same buffer they were assembled with. This image was captured on a scale of 50 nm at 50,000x magnification using the Biological Electron Microscopy Facility at the University of Hawaii at Mānoa. Many capsid intermediates were observed, but no VLPs.

![](https://static.igem.org/mediawiki/2018/c/c7/T--Hawaii--exp_fig27.jpg)

**Figure 5.** Construct 3a with a His-tag using an assembly buffer with basic pH. VLPs were imaged in the same buffer they were assembled with. This image was captured on a scale of 20 nm at 80,000x magnification using the Biological Electron Microscopy Facility at the University of Hawaii at Mānoa.

![](https://static.igem.org/mediawiki/2018/3/3a/T--Hawaii--exp_fig28.jpg)

**Figure 6.** Construct 3a with no His-tag using an assembly buffer with a pH of 7.3. VLPs were imaged in the same buffer they were assembled with. This image was captured on a scale of 20 nm at 100,000x magnification using the Biological Electron Microscopy Facility here at the University of Hawaii at Mānoa. VLP diameter is measured at 35.4 nm.

### III. Assembly of the Purified Proteins Constructs and Assembly Time Assays

**Purpose:** To assemble all other protein constructs into VLP’s using the standard VLP assembly buffer and to observe VLP assembly at different stages of time. Construct 3a was also purified and assembled under acidic and basic conditions to confirm the results from the first VLP experiment.

**Materials and Methods:** Construct 1 (HisTEV-CA-NC) insoluble proteins were purified into acidic VLP assembly buffer, and then some proteins were separated for His-tag cleavage. Construct 2a (His-TEV-CA1) was also purified and had it’s his-tag cleaved. Construct 3a proteins taken from the last protein purification stock its VLPs were assembled under acidic and basic conditions. During the water bath incubation, Construct 1 VLPs were removed at 1 hour, 2 hours, and 3 hours. Only 100 ng/ul of protein was used for all assemblies. All VLP samples were observed under the TEM at the University of Hawaii at Manoa.

**Results and Discussion:** Many of the samples observed under the EM showed little to no VLPs suggesting that something had gone wrong during the assembly process. This suggests that the full gag construct may not react well in an acidic assembly buffer, however there were very little VLPs observed for Construct 3a assembled at acidic conditions; a protein that has already been proven to assemble under acidic conditions. However, some VLPs were observed in the Construct 1 assemblies (Figure 1-3). This experiment would have to be repeated in order to obtain more reliable results. Construct 1 VLP assembly may have to be tested under neutral or basic conditions to verify optimal assembly conditions for the full gag, or more protein should be used for the VLP assembly instead of 100 ng. Construct 2a, which consists of a capsid protein of about 37 kDa in size, did not reveal any VLP’s under the EM (Figure 4). Since the capsid for construct 3a also did not assemble as it should have, it is reasonable to assume that the assembly reaction may have prohibited by some confounding factor.

![](https://2018.igem.org/File:T--Hawaii--exp_fig29.jpg)

**Figure 1.** A potential VLP that was assembled in only 1 hour. This image was captured on a scale of 20 nm at 100,000x magnification using the Biological Electron Microscopy Facility here at the University of Hawaii at Mānoa.

![](https://static.igem.org/mediawiki/2018/b/b1/T--Hawaii--exp_fig30.jpg)

**Figure 2.** Some potential VLPs were observed for untagged construct 1 assembled at 2 hours.This image was captured on a scale of 20 nm at 70,000x magnification using the Biological Electron Microscopy Facility here at the University of Hawaii at Mānoa.

![](https://static.igem.org/mediawiki/2018/8/80/T--Hawaii--exp_fig31.jpg)

**Figure 3.** Mostly VLP intermediates were observed for untagged construct 1 assembled at 3 hours. No possible VLPs were identified.This image was captured on a scale of 20 nm at 70,000x magnification using the Biological Electron Microscopy Facility here at the University of Hawaii at Mānoa. No possible VLPs were identified.

## REFERENCES

* [1] Parent, L. J., & Gudleski, N. (2011). Beyond Plasma Membrane Targeting: Role of the MA domain of Gag in Retroviral Genome Encapsidation. Journal of Molecular Biology, 410(4), 553–564.
* [2] Abdusetir Cerfoglio, J. C., González, S. A., & Affranchino, J. L. (2014). Structural elements in the Gag polyprotein of feline immunodeficiency virus involved in Gag self-association and assembly. Journal of General Virology, 95(9), 2050–2059.
* [3] Syomin, B. V., Leonova, O. G., Trendeleva, T. A., Zvyagilskaya, R. A., Ilyin, Y. V., & Popenko, V. I. (2012). Effect of nucleocapsid on multimerization of structural protein GAG. Molecular Biology, 46(2), 270–278.
* [4] Goh, B. C., Perilla, J. R., England, M. R., Heyrana, K. J., Craven, R. C., & Schulten, K. (2015). Atomic Modeling of an Immature Retroviral Lattice using Molecular Dynamics and Mutagenesis. Structure (London, England : 1993), 23(8), 1414–1425.
* [5] Kuznetsov, Yurii G., Zhang, Min, Menees, Thomas M., McPherson, Alexander, & Sandmeyer, Suzanne. (2005). Investigation by Atomic Force Microscopy of the Structure of Ty3 Retrotransposon Particles. The Journal of Virology, 79(13), 8032-45.
* [6] Roth, J.-F. (2000). The yeast Ty virus-like particles. Yeast, 16(9), 785–795.
* [7] Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A.;Protein Identification and Analysis Tools on the ExPASy Server.
* [8] Podell, Sheila, and Michael Gribskov. “Predicting N-Terminal Myristoylation Sites in Plant Proteins.” BMC Genomics 5 (2004): 37. PMC. Web. 18 Oct. 2018.
* [9] Christine L. Nguyen, Karl Münger Journal of Virology Jan 2009, 83 (4) 1700-1707.
* [10] Kosugi et al., Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proceedings of the National Academy of Sciences Aug 2009, 106 (31) 13142.
* [11] Lin, S. S., Nymark-McMahon, M. H., Yieh, L., & Sandmeyer, S. B. (2001). Integrase Mediates Nuclear Localization of Ty3. Molecular and Cellular Biology, 21(22), 7826–7838.
* [12] Zhang, W., Lee, H.-R., Koo, D.-H., & Jiang, J. (2008). Epigenetic Modification of Centromeric Chromatin: Hypomethylation of DNA Sequences in the CENH3-Associated Chromatin in Arabidopsis thaliana and Maize. The Plant Cell Online, 20(1), 25–34.
* [13] Jiang, J., Birchler, J. A., Parrott, W. A., & Kelly Dawe, R. (2003). A molecular view of plant centromeres. Trends in Plant Science, 8(12), 570–575.
* [14] Melo, Devos, Depiereux, & Feytmans. (1997). ANOLEA: A www server to assess protein structures. Proceedings. International Conference on Intelligent Systems for Molecular Biology, 5, 187-90.
* [15] R. Lüthy, J.U. Bowie, D. Eisenberg. Assessment of protein models with three-dimensional profiles Nature, 356 (1992), pp. 83-85
* [16] Berman, H., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T., Weissig, H., . . . Bourne, P. (2000). The Protein Data Bank. Nucleic Acids Research,28(1), 235-42.
* [17] Haider, S. R., Reid, H. J., & Sharp, B. L. (2012). Tricine-SDS-PAGE. In B. T. Kurien & R. H. Scofield (Eds.), Protein Electrophoresis (Vol. 869, pp. 81–91). Totowa, NJ: Humana Press.

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#### AUTOCATALYTIC PROTEASE

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#### AUTOCATALYTIC PROTEASE

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Lorem ipsum dolor sit amet, consectetur adipisicing elit. Fugiat repellendus ipsam a odit soluta eveniet id pariatur voluptatem odio. Necessitatibus facere optio quaerat magni ipsa esse nam minima voluptatum quos.

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#### AUTOCATALYTIC PROTEASE

May 5, 2018

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#### AUTOCATALYTIC PROTEASE

May 5, 2018

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Fugiat repellendus ipsam a odit soluta eveniet id pariatur voluptatem odio. Necessitatibus facere optio quaerat magni ipsa esse nam minima voluptatum quos.

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View Experiments

![](https://media-cdn.tripadvisor.com/media/photo-s/11/49/24/7f/omen-sooty-owl.jpg)

#### AUTOCATALYTIC PROTEASE

May 5, 2018

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Fugiat repellendus ipsam a odit soluta eveniet id pariatur voluptatem odio. Necessitatibus facere optio quaerat magni ipsa esse nam minima voluptatum quos.

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Lorem ipsum dolor sit amet, consectetur adipisicing elit. Fugiat repellendus ipsam a odit soluta eveniet id pariatur voluptatem odio. Necessitatibus facere optio quaerat magni ipsa esse nam minima voluptatum quos.

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Fugiat repellendus ipsam a odit soluta eveniet id pariatur voluptatem odio. Necessitatibus facere optio quaerat magni ipsa esse nam minima voluptatum quos.

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View Experiments

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#### AUTOCATALYTIC PROTEASE

May 5, 2018

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Fugiat repellendus ipsam a odit soluta eveniet id pariatur voluptatem odio. Necessitatibus facere optio quaerat magni ipsa esse nam minima voluptatum quos.

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Lorem ipsum dolor sit amet, consectetur adipisicing elit. Fugiat repellendus ipsam a odit soluta eveniet id pariatur voluptatem odio. Necessitatibus facere optio quaerat magni ipsa esse nam minima voluptatum quos.

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## CONTACT

uhigem@hawaii.edu

## LOCATE

University of Hawaii at Manoa

1955 East West Road

Honolulu, HI 96822

## NAVIGATE

Home
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